Prostate specific genes and the use thereof as targets for prostate cancer therapy

ABSTRACT

Genes that are upregulated in human prostate tumor tissues and the corresponding proteins are identified. These genes and the corresponding antigens are suitable targets for the treatment, diagnosis or prophylaxis of prostate cancer.

FIELD OF THE INVENTION

The present invention relates to the identification of DNA sequencesthat correspond to alternatively spliced events in genes expressed onthe surface of prostate cancer cells. These genes or their correspondingproteins are to be targeted for the treatment, prevention and/ordiagnosis of cancers wherein these genes are differentially regulatedand/or spliced, particularly in prostate cancer.

BACKGROUND OF THE INVENTION

Genetic detection of human disease states is a rapidly developing field(Taparowsky et al., 1982; Slamon et al., 1989; Sidransky et al., 1992;Miki et al., 1994; Dong et al., 1995; Morahan et al., 1996; Lifton,1996; Barinaga, 1996). However, some problems exist with this approach.A number of known genetic lesions merely predispose an individual to thedevelopment of specific disease states. Individuals carrying the geneticlesion may not develop the disease state, while other individuals maydevelop the disease state without possessing a particular geneticlesion. In human cancers, genetic defects may potentially occur in alarge number of known tumor suppresser genes and proto-oncogenes.

Genetic detection of cancer has a long history. Some of the earliestgenetic lesions shown to predispose to cancer were transforming pointmutations in the ras oncogenes (Taparowsky et al., 1982). Transformingras point mutations may be detected in the stool of individuals withbenign and malignant colorectal tumors (Sidransky et al., 1992).However, only 50% of such tumors contained a ras mutation (Sidransky etal., 1992). Similar results have been obtained with amplification ofHER-2/neu in breast and prostate cancer (Slamon et al., 1989), deletionand mutation of p53 in bladder cancer (Sidransky et al., 1991), deletionof DCC in colorectal cancer (Fearon et al., 1990) and mutation of BRCA1in breast and prostate cancer (Miki et al., 1994).

None of these genetic lesions are capable of predicting a majority ofindividuals with cancer and most require direct sampling of a suspectedtumor, and make screening difficult. Further, none of the markersdescribed above are capable of distinguishing between metastatic andnon-metastatic forms of cancer. In effective management of cancerpatients, identification of those individuals whose tumors have alreadymetastasized or are likely to metastasize is critical. Becausemetastatic cancer kills 560,000 people in the U.S. each year (ACS homepage), identification of markers for metastatic prostate cancer would bean important advance.

A particular problem in cancer detection and diagnosis occurs withprostate cancer. Carcinoma of the prostate is the most frequentlydiagnosed cancer among men in the United States (Veltri et al., 1996).Prostate cancer was diagnosed in approximately 189,500 men in 1998 andabout 40,000 men succumbed to the malignancy (Landis et al, 1998).Although relatively few prostate tumors progress to clinicalsignificance during the lifetime of the patient, those which areprogressive in nature are likely to have metastasized by the time ofdetection. Survival rates for individuals with metastatic prostatecancer are quite low. Between these extremes are patients with prostatetumors that will metastasize but have not yet done so, for whom surgicalprostate removal is curative. Determination of which group a patientfalls within is critical in determining optimal treatment and patientsurvival.

The FDA approval of the serum prostate specific antigen (PSA) test in1984 changed the way that prostate disease was managed (Allhoff et al.,1989; Cooner et al., 1990; Jacobson et al, 1995; Orozco et al., 1998).PSA is widely used as a serum biomarker to detect and monitortherapeutic response in prostate cancer patients (Badalament et al.,1996; O'Dowd et al., 1997). Several modifications in PSA assays (Partinand Oesterling, 1994; Babian et al., 1996; Zlotta et al, 1997) haveresulted in earlier diagnoses and improved treatment.

Although PSA has been widely used as a clinical marker of prostatecancer since 1988 (Partin and Oesterling, 1994), screening programsutilizing PSA alone or in combination with digital rectal examination(DRE) have not been successful in improving the survival rate for menwith prostate cancer (Partin and Oesterling, 1994). Although PSA isspecific to prostate tissue, it is produced by normal and benign as wellas malignant prostatic epithelium, resulting in a high false-positiverate for prostate cancer detection (Partin and Oesterling, 1994).

While an effective indicator of prostate cancer when serum levels arerelatively high, PSA serum levels are more ambiguous indicators ofprostate cancer when only modestly elevated, for example when levels arebetween 2-10 ng/ml. At these modest elevations, serum PSA may haveoriginated from non-cancerous disease states such as BPH (benignprostatic hyperplasia), prostatitis or physical trauma (McCormack et al,1995). Although application of the lower 2.0 ng/ml cancer detectioncutoff concentration of serum PSA has increased the diagnosis ofprostate cancer, especially in younger men with nonpalpable early stagetumors (Stage Tlc) (Soh et al., 1997; Carter and Coffey, 1997; Harris etal., 1997; Orozco et al., 1998), the specificity of the PSA assay forprostate cancer detection at low serum PSA levels remains a problem.

Several investigators have sought to improve upon the specificity ofserologic detection of prostate cancer by examining a variety of otherbiomarkers besides serum PSA concentration (Ralph and Veltri, 1997). Oneof the most heavily investigated of these other biomarkers is the ratioof free versus total PSA (f/t PSA) in a patient's blood. Most PSA inserum is in a molecular form that is bound to other proteins such asalpha1-antichymotrypsin (ACT) or alpha2-macroglobulin (Christensson etal, 1993; Stenman et al., 1991; Lilja et al., 1991). Free PSA is notbound to other proteins. The ratio of free to total PSA (f/tPSA) isusually significantly higher in patients with BPH compared to those withorgan confined prostate cancer (Marley et al., 1996; Oesterling et al.,1995; Pettersson et al., 1995). When an appropriate cutoff is determinedfor the f/tPSA assay, the f/tPSA assay can help distinguish patientswith BPH from those with prostate cancer in cases in which serum PSAlevels are only modestly elevated (Marley et al., 1996; Partin andOesterling, 1996). Unfortunately, while f/tPSA may improve on thedetection of prostate cancer, information in the f/tPSA ratio isinsufficient to improve the sensitivity and specificity of serologicdetection of prostate cancer to desirable levels.

Other markers that have been used for prostate cancer detection includeprostatic acid phosphatase (PAP) and prostate secreted protein (PSP).PAP is secreted by prostate cells under hormonal control (Brawn et al.,1996). It has less specificity and sensitivity than does PSA. As aresult, it is used much less now, although PAP may still have someapplications for monitoring metastatic patients that have failed primarytreatments. In general, PSP is a more sensitive biomarker than PAP, butis not as sensitive as PSA (Huang et al., 1993). Like PSA, PSP levelsare frequently elevated in patients with BPH as well as those withprostate cancer.

Another serum marker associated with prostate disease is prostatespecific membrane antigen (PSMA) (Horoszewicz et al., 1987; Carter andCoffey, 1996; Murphy et al., 1996). PSMA is a Type II cell membraneprotein and has been identified as Folic Acid Hydrolase (FAH) (Carterand Coffey, 1996). Antibodies against PSMA react with both normalprostate tissue and prostate cancer tissue (Horoszewicz et al., 1987).Murphy et al. (1995) used ELISA to detect serum PSMA in advancedprostate cancer. As a serum test, PSMA levels are a relatively poorindicator of prostate cancer. However, PSMA may have utility in certaincircumstances. PSMA is expressed in metastatic prostate tumor capillarybeds (Silver et al., 1997) and is reported to be more abundant in theblood of metastatic cancer patients (Murphy et al., 1996). PSMAmessenger RNA (mRNA) is down-regulated 8-10 fold in the LNCaP prostatecancer cell line after exposure to 5-alpha-dihydroxytestosterone (DHT)(Israeli et al., 1994).

Two relatively new potential biomarkers for prostate cancer are humankallekrein 2 (HK2) (Piironen et al., 1996) and prostate specifictransglutaminase (pTGase) (Dubbink et al., 1996). HK2 is a member of thekallekrein family that is secreted by the prostate gland (Piironen etal., 1996). Prostate specific transglutaminase is a calcium-dependentenzyme expressed in prostate cells that catalyzes post-translationalcross-linking of proteins (Dubbink et al., 1996). In theory, serumconcentrations of HK2 or pTGase maybe of utility in prostate cancerdetection or diagnosis, but the usefulness of these markers is stillbeing evaluated.

Interleukin 8 (IL-8) has also been reported as a marker for prostatecancer. (Veltri et al., 1999). Serum IL-8 concentrations were reportedto be correlated with increasing stage of prostate cancer and to becapable of differentiating BPH from malignant prostate tumors. (Id.) Thewide-scale applicability of this marker for prostate cancer detectionand diagnosis is still under investigation.

In addition to these protein markers for prostate cancer, severalgenetic changes have been reported to be associated with prostatecancer, including: allelic loss (Bova, et al., 1993; Macoska et al.,1994; Carter et al., 1990); DNA hypermethylation (Isaacs et al., 1994);point mutations or deletions of the retinoblastoma (Rb), p53 and KAI1genes (Bookstein et al., 1990a; Bookstein et al., 1990b; Isaacs et al.,1991; Dong et al., 1995); and aneuploidy and aneusomy of chromosomesdetected by fluorescence in situ hybridization (FISH) (Macoska et al.,1994; Visakorpi et al., 1994; Takahashi et al., 1994; Alcaraz et al.,1994). None of these have been reported to exhibit sufficientsensitivity and specificity to be useful as general screening tools forasymptomatic prostate cancer.

In current clinical practice, the serum PSA assay and digital rectalexam (DRE) is used to indicate which patients should have a prostatebiopsy (Lithrup et al., 1994; Orozco et al., 1998). Histologicalexamination of the biopsied tissue is used to make the diagnosis ofprostate cancer. Based upon the 189,500 cases of diagnosed prostatecancer in 1998 (Landis, 1998) and a known cancer detection rate of about35% (Parker et al., 1996), it is estimated that in 1998 over one-halfmillion prostate biopsies were performed in the United States (Orozco etal., 1998; Veltri et al., 1998). Clearly, there would be much benefitderived from a serological test that was sensitive enough to detectsmall and early stage prostate tumors that also had sufficientspecificity to exclude a greater portion of patients with noncancerousor clinically insignificant conditions.

There remain deficiencies in the prior art with respect to theidentification of the genes linked with the progression of prostatecancer and the development of diagnostic methods to monitor diseaseprogression. Likewise, the identification of genes, which aredifferentially expressed in prostate cancer, would be of considerableimportance in the development of a rapid, inexpensive method to diagnosecancer. Although a few prostate specific genes have been cloned (PSA,PSMA, HK2, pTGase, etc.), these are typically not upregulated inprostate cancer. The identification of a novel, prostate specific genethat is differentially expressed in prostate cancer, compared tonon-malignant prostate tissue, would represent a major, unexpectedadvance for the diagnosis, prognosis and treatment of prostate cancer.

The use of therapeutic antibodies for treatment of cancers that targetsurface proteins is known. Examples thereof include RITUXAN® thattargets CD20 on B cell lymphoma, Campath® that targets a surface antigenCD52 expressed by chronic lymphocytic leukemia, Herceptin® that targetserbB2 on breast and other cancers and Mybtara that targets CD33 surfaceantigen expressed on leukemia cells. However, to date, a monoclonalantibody for treatment of prostate cancer has not been approved fortherapeutic use.

SUMMARY OF THE INVENTION

The present invention relates to the identification of novel nucleicacid and amino acid sequences that are characteristic of prostate cancercell or tissue, and which represent targets for therapy or diagnosis ofsuch a condition in a subject.

The invention more specifically discloses 159 specific, isolated nucleicacid molecules that encode novel expression sequences. Of these, 122 areexpressed sequence tags that are differentially spliced and correspondto SEQ ID NOS 1-65, 74, 80, 85, 102-134, 136, 141, 146, 150-165, 167,168. In addition, 42 specific isoforms of known genes have beenidentified corresponding to SEQ ID NOS. 67-72, 75-77, 81-83, 86-90, 92,93, 95-98, 100, 101, 137-139, 143, 144, 147-149, 169-173, 175, 177, 179,and 181. These novel sequences were found to be differentially expressedbetween normal prostate and prostate cancer. The expressed sequence tagrepresent novel exons that are alternatively spliced in prostate cancer,and as such, directly identify distinct isoforms. These sequences andmolecules represent targets and valuable information to develop methodsand materials for the detection, diagnosis, and treatment of prostatecancer.

It is an object of the invention to provide methods and materials fortreatment and diagnosis of prostate cancer.

It is a more specific object of the invention to identify novel exons(novel splice variants) that are expressed by prostate cancer tissuewhich are potential gene targets for treatment and diagnosis of prostatecancer.

It is a specific object of the invention to develop novel therapies fortreatment of prostate cancer involving the administration or use ofanti-sense oligonucleotides corresponding to novel gene targets that arespecifically expressed by the prostate cancer.

It is another specific object of the invention to identify exons and thecorresponding protein domain encoded by those exons specificallyupregulated in prostate cancer cells.

It is another specific object of the invention to produce ligands thatbind antigens encoded by the exons, expressed as a protein domain bycertain prostate cancers, including, but not limited to, monoclonalantibodies.

It is another specific object of the invention to provide noveltherapeutic regimens for the treatment of prostate cancer that involvethe administration or use of antigens expressed by certain prostatecancers, alone or in combination with adjuvants that elicit anantigen-specific cytotoxic T-cell lymphocyte response against cancercells that express such antigen.

It is another object of the invention to provide novel therapeuticregimens for the treatment of prostate cancer that involve theadministration or use of ligands, especially monoclonal antibodies thatspecifically bind novel antigens that are expressed by certain prostatecancers.

It is an other object of this invention to provide pharmaceuticalcompositions comprising a ligand or antigen as defined above, incombination with a pharmaceutically acceptable carrier or excipientand/or an adjuvant.

It is another object of the invention to provide a novel method fordiagnosis of prostate cancer by using ligands, e.g., monoclonalantibodies, which specifically bind to antigens that are specificallyexpressed by certain prostate cancers, in order to detect whether asubject has or is at increased risk of developing prostate cancer.

It is another object of the invention to provide a novel method ofdetecting persons having, or at increased risk of developing prostatecancer by use of labeled DNAs that hybridize to novel gene targetsexpressed by certain prostate cancers.

It is yet another object of the invention to provide diagnostic testkits for the detection of persons having or at increased risk ofdeveloping prostate cancer that comprise a ligand, e.g., monoclonalantibody that specifically binds to an antigen expressed by prostatecancer cells, and a detectable label, e.g. indicator enzymes, aradiolabels, fluorophores, or paramagnetic particles.

It is another object of the invention to provide diagnostic kits fordetection of persons having or at risk of developing prostate cancerthat comprise DNA primers or probes specific for novel gene targetsspecifically expressed by prostate cancer cells, and a detectable label,e.g. indicator enzymes, a radiolabels, fluorophores, or paramagneticparticles.

It is another object of this invention to provide methods for selecting,identifying, screening, characterizing or optimizing biologically activecompounds, comprising a determination of whether a candidate compoundbinds, preferably selectively, an antigen or a polynucleotide asdisclosed in the present application. Such compounds represent drugcandidates or leads for treating cancer diseases, particularly prostatecancer.

It is another object of the invention to identify genes that areexpressed in altered forms in prostate cancer cells. These formsrepresent splice variants of the gene, where the DATAS™ fragmenteither 1) indicates the splice event occurring within the gene, or 2)points to a gene that is actively spliced to produce different geneproducts. These different splice variants or isoforms can be targets fortherapeutic intervention.

LEGEND TO THE FIGURES

FIG. 1: Expression of Sequence ID: No. 92 in normal human tissue.Primers were designed to detect the DATAS clone sequence and RT-PCRanalysis was performed for 30 cycles. Lane 1, Prostate; lane 2, Heart;lane 3, Lung; lane 4, Kidney; lane 5, Liver; lane 6, Brain; lane 7,Placenta; lane 8, Sk. Muscle; lane 9, Pancreas; lane 10, Spleen; lane11, Thymus; lane 12, Testis; lane 13, Ovary; lane 14, Sm. Intestine;lane 15, Colon; lane 16 Leukocyte.

FIG. 2: Expression of clone (SEQ ID NO 92) in normal and tumor prostatesamples. Primers were designed to detect the DATAS clone and RT-PCRanalysis was performed for 40 cycles. Individual RNA samples (normal andtumor) were tested both as pooled and as individual samples. The pooledRNA samples were used to produce cDNA using either an oligo dT approach(dT) or through a random primer protocol (RP). Individual patient cDNAsamples (lanes 9-12) were prepared through the random primed protocol.Lane 1, prostate tumor pool 1 (RP cDNA); lane 2, normal prostate pool 1(RP cDNA); lane 3, prostate tumor pool 2 (RP cDNA); lane 4, normalprostate pool 2 (RP cDNA); lane 5, prostate tumor pool 1 (dT cDNA); lane6, normal prostate pool ¹ (dT cDNA); lane 7, normal prostate pool 2 (dTcDNA); lane 8, NTC; lane 9, Patient 1 (OHK); lane 10, Patient 2 (T523);lane 11, Patient 3 (82B) ; lane 12, Patient 4 (4BK).

FIG. 3: Alignment of the different isoforms isolated from structuralanalysis of clone (DATAS clone number). The sequences isolated from theDATAS derived events were mapped using Blat against the Human genome toannotate the gene and determine the each unique splicing event. Fiveevents are mapped with AK092666, an EST that closely resembles the fiveevents.

FIG. 4: Western blot analysis for the expression of STEAP2 isoforms.Protein extracts from prostate cancer cell lines were separated onSDS_PAGE gels and transferred to nitrocellulose, and probed with anantibody raised against a peptide sequence present in the N-terminalportion of the wild type STEAP2 protein. Five different cell lines wereanalyzed: lane 1) LNCaP; 2) 22Rv1 3) MDA-PCa2b; 4) PC3; 5) DU145. Theblot was developed using standard chemilumninescence reagents.

DETAILED DESCRIPTION OF THE INVENTION

DATAS (Different Analysis of Transcripts with Alternative Splicing)analyzes structural differences between expressed genes and providessystematic access to alterations in RNA splicing (disclosed in U.S. Pat.No.6,251,590, the disclosure of which is incorporated by reference inits entirety). Having access to these spliced sequences, which arecritical for cellular homeostasis, represents a useful advance infunctional genomics.

The DATAS Technology generates two libraries when comparing two samples,such as normal vs. tumor tissue. Each library specifically containsclones of sequences that are present and more highly expressed in onesample. For example, library A will contain sequences that are presentin genes in the normal samples but absent in the tumor samples. Thesesequences are identified as being removed or spliced out from the genesin the tumor samples. In contrast, library B will contain sequences thatare present only in the tumor samples and not present in the normalsamples. These represent exons/introns that are alternatively splicedinto genes expressed only in the tumor samples.

The present invention is based in part on the identification of exonsthat are isolated using DATAS and then determined to be differentiallyregulated or expressed in prostate tumor samples. Specifically, 122expressed sequence tags were identified through DATAS and confirmed tobe differentially expressed between normal prostate tissue and prostatetumor tissue. These DATAS fragments (DF) are small sections of genesthat are selected for inclusion or exclusion in one sample but not theother. These small sections are part of the expressed gene transcript,and can consist of sequences derived from several different regions ofthe gene, including, but not limited to, portions of single exons,several exons, sequence from introns, and sequences from exons andintrons. This alternative usage of exons in different biological samplesproduces different gene products from the same gene through a processwell known in the art as alternative RNA splicing. In particular, 37alternatively spliced isoforms have been identified from the DATASfragment sequences, and produce alternate gene products that fit all thedescriptions of targets and gene products below.

Alternatively spliced mRNA's produced from the same gene containdifferent ribonucleotide sequence, and therefore translate into proteinswith different amino acid sequences. Nucleic acid sequences that arealternatively spliced into or out of the gene products can be insertedor deleted in frame or out of frame from the original gene sequence.This leads to the translation of different proteins from each variant.Differences can include simple sequence deletions, or novel sequenceinformation inserted into the gene product. Sequences inserted out offrame can lead to the production of an early stop codon and produce atruncated form of the protein. Alternatively, in-frame insertions ofnucleic acid may cause an additional protein domain to be expressed fromthe mRNA. The end stage target is a novel protein containing either anovel epitope or function. Many variations of known genes have beenidentified and produce protein variants that can be agonistic orantagonistic with the original biological activity of the protein.

DATAS fragments thus identify genes and proteins which are subject todifferential regulation and alternative splicing(s) in prostate cancercells. DATAS fragments thus allow the definition of target moleculessuitable for diagnosis or therapy of prostate cancers, which targetmolecules comprise all or a portion of genes or RNAs comprising thesequence of a DATAS fragment, or of genes or RNA from which the sequenceof a DATAS fragment derives, as well as corresponding polypeptides orproteins, and variants thereof.

A first type of target molecule is a target nucleic acid moleculecomprising the sequence of a full gene or RNA molecule comprising thesequence of a DATAS fragment as disclosed in the present application.Indeed, since DATAS identifies genetic deregulations associated withprostate tumor, the whole gene or RNA sequence from which said DATASfragment derives can be used as a target of therapeutic intervention ordiagnosis.

Similarly, another type of target molecule is a target polypeptidemolecule comprising the sequence of a full-length protein comprising theamino acid sequence encoded by a DATAS fragment as disclosed in thepresent application.

A further type of target molecule is a target nucleic acid moleculecomprising a fragment of a gene or RNA as disclosed above. Indeed, sinceDATAS identifies genes and RNAs that are altered in prostate tumorcells, portions of such genes or RNAs, including portions that do notcomprise the sequence of a DATAS fragment, can be used as a target fortherapeutic intervention or diagnosis. Examples of such portions includeDATAS fragments, portions thereof, alternative exons or introns of saidgene or RNA, exon-exon, exon-intron or intron-intron junction sequencesgenerated by splicing(s) in said RNA, etc. Particular portions comprisea sequence encoding a extra-cellular domain of a polypeptide.

Similarly, another type of target molecule is a fragment of a proteincomprising the amino acid sequence encoded by a DATAS fragment asdisclosed in the present application. Such fragments may comprise or notthe DATAS sequence, and may comprise newly generated amino acid sequenceresulting, for instance, from a frame shift, a novel exon-exon orexon-intron junction, the creation of new stop codon, etc.

These target molecules (including genes, fragments, proteins and theirvariants) can serve as diagnostic agents and as targets for thedevelopment of therapeutics. For example, these therapeutics maymodulate biological processes associated with prostate tumor viability.Agents may also be identified that are associated with the induction ofapoptosis (cell death) in prostate tumor cells. Other agents can also bedeveloped, such as monoclonal antibodies, that bind to the protein orits variant and alter the biological processes important for cellgrowth. Alternatively, antibodies can deliver a toxin which can inhibitcell growth and lead to cell death.

Specifically, the invention provides sequences that are expressed in avariant protein and are prostate tumor specific or prostate specific.These sequences are portions of genes identified to be in the plasmamembrane of the cell through bioinformatic analysis, and the specificsequences of the invention are expressed on the extracellular region ofthe protein, so that the sequences may be useful in the preparation ofprostate tumor vaccines, including prophylatic and therapeutic vaccines.

Based thereon, it is anticipated that the disclosed genes that areassociated with the differentially expressed sequences and thecorresponding variant proteins should be suitable targets for prostatecancer therapy, prevention or diagnosis, e.g. for the development ofantibodies, small molecular inhibitors, anti-sense therapeutics, andribozymes. The potential therapies are described in greater detailbelow.

Such therapies will include the synthesis of oligonucleotides havingsequences in the antisense orientation relative to the subject nucleicacids which appear to be up-regulated in prostate cancer. Suitabletherapeutic antisense oligonucleotides will typically vary in lengthfrom two to several hundred nucleotides in length, more typically about50-70 nucleotides in length or shorter. These antisense oligonucleotidesmay be administered as naked nucleic acids or in protected forms, e.g.,encapsulated in liposomes. The use of liposomal or other protected formsmay be advantageous as it may enhance in vivo stability and thusfacilitate delivery to target sites, i.e., prostate tumor cells.

Also, the subject novel genes may be used to design novel ribozymes thattarget the cleavage of the corresponding mRNAs in prostate tumor cells.Similarly, these ribozymes may be administered in free (naked) form orby the use of delivery systems that enhance stability and/or targeting,e.g., liposomes.

Also, the present invention embraces the administration of use ofnucleic acids that hybridize to the novel nucleic acid targetsidentified infra, attached to therapeutic effector moieties, e.g.,radiolabels, (e.g., ⁹⁰Y, ¹³¹I) cytotoxins, cytotoxic enzymes, and thelike in order to selectively target and kill cells that express thesenucleic acids, i.e., prostate tumor cells.

Also, the present invention embraces the treatment and/or diagnosis ofprostate cancer by targeting altered genes or the corresponding alteredprotein particularly splice variants that are expressed in altered formin prostate tumor cells. These methods will provide for the selectivedetection of cells and/or eradication of cells that express such alteredforms thereby minimizing adverse effects to normal cells.

Still further, the present invention encompasses non-nucleic acid basedtherapies. For example, the invention encompasses the use of a DNAcontaining one of the novel cDNAs corresponding to novel antigenidentified herein. It is anticipated that the antigens so encoded may beused as therapeutic or prophylactic anti-tumor vaccines. For example, aparticular contemplated application of these antigens involves theiradministration with adjuvants that induce a cytotoxic T lymphocyteresponse.

Administration of the subject novel antigens in combination with anadjuvant may result in a humoral immune response against such antigens,thereby delaying or preventing the development of prostate cancer.

These embodiments of the invention will comprise administration of oneor more of the subject novel prostate cancer antigens, ideally incombination with an adjuvant, e.g., PROVAX™ (as disclosed U.S. Pat. Nos.5,709,860, 5,695,770, and 5,585,103, which comprises a microfluidizedadjuvant containing Squalene, Tween and Pluronic), ISCOM'S®, DETOX®,SAF, Freund's adjuvant, Alum®, Saponin®, among others. This compositionwill be administered in an amount sufficient to be therapeutically orprophylactically effective, e.g. on the order of 50 to 20,000 mg/kg bodyweight, 100 to 5000 mg/kg body weight.

Yet another embodiment of the invention will comprise the preparation ofmonoclonal antibodies against the antigens encoded by the novel genescontaining the nucleic acid sequences disclosed infra. Such monoclonalantibodies may be produced by conventional methods and include humanmonoclonal antibodies, humanized monoclonal antibodies, chimericmonoclonal antibodies, single chain antibodies, e.g., scFv's andantigen-binding antibody fragments such as Fab and Fab′ fragments.Methods for the preparation of monoclonal antibodies are known in theart. In general, preparation of monoclonal antibodies will compriseimmunization of an appropriate (non-homologous) host with the subjectprostate cancer antigens, isolation of immune cells therefrom, use ofsuch immune cells to isolate monoclonal antibodies and screening formonoclonal antibodies that specifically bind to either of such antigens.Antibody fragments maybe prepared by known methods, e.g., enzymaticchange of monoclonal antibodies.

These monoclonal antibodies and fragments will be useful for passiveanti-tumor immunotherapy, or may be attached to therapeutic effectormoieties, e.g., radiolabels, cytotoxins, therapeutic enzymes, agentsthat induce apoptosis, and the like in order to provide for targetedcytotoxicity, i.e., killing of human prostate tumor cells. Given thefact that the subject genes are apparently not significantly expressedby many normal tissues this should not result in significant adverseside effects (toxicity to non-target tissues).

In one embodiment, of the present invention such antibodies or fragmentswill be administered in labeled or unlabeled form, alone or inconjunction with other therapeutics, e.g., chemotherapeutics such ascisplatin, methotrexate, adriamycin, and the like suitable for prostatecancer therapy. The administered composition will also typically includea pharmaceutically acceptable carrier, and optionally adjuvants,stabilizers, etc., used in antibody compositions for therapeutic use.

Preferably, the subject monoclonal antibodies will bind the targetantigens with high affinity, e.g., possess a binding affinity (Kd) onthe order of 10⁻⁶ to 10⁻¹² M.

As noted, the present invention also embraces diagnostic applicationsthat provide for detection of the expression of prostate specific splicevariants disclosed herein. This will comprise detecting the expressionof one or more of these genes at the RNA level and/or at the proteinlevel.

For nucleic acids, expression of the subject genes will be detected byknown nucleic acid detection methods, e.g., Northern blot hybridization,strand displacement amplification (SDA), catalytic hybridizationamplification (CHA), and other known nucleic acid detection methods.Preferably, a cDNA library will be made from prostate cells obtainedfrom a subject to be tested for prostate cancer by PCR using primerscorresponding to the novel isoforms disclosed in this application.

The presence or absence of prostate cancer can be determined based onwhether PCR products are obtained, and the level of expression. Thelevels of expression of such PCR product may be quantified in order todetermine the prognosis of a particular prostate cancer patient (as thelevels of expression of the PCR product often will increase or decreasesignificantly as the disease progresses.) This may provide a method formonitoring the status of a prostate cancer patient.

Alternatively, the status of a subject to be tested for prostate cancermay be evaluated by testing biological fluids, e.g., blood, urine,lymph, and the like with an antibody or antibodies or fragment thatspecifically binds to the novel prostate tumor antigens disclosedherein.

Methods for using antibodies to detect antigen expression are well knownand include ELISA, competitive binding assays, and the like. In general,such assays use an antibody or antibody fragment that specifically bindsthe target antigen directly or indirectly bound to a label that providesfor detection, e.g. indicator enzymes, a radiolabels, fluorophores, orparamagnetic particles.

Patients which test positive for the enhanced presence of the antigen onprostate cells will be diagnosed as having or being at increased risk ofdeveloping prostate cancer. Additionally, the levels of antigenexpression may be useful in determining patient status, i.e., how fardisease has advanced (stage of prostate cancer).

As noted, the present invention provides novel splice variants thatencode antigens that correlate to human prostate cancer. The presentinvention also embraces variants thereof. As used herein “variants”means sequences that are at least about 75% identical thereto, morepreferably at least about 85% identical, and most preferably at least90% identical and still more preferably at least about 95-99% identifiedwhen these DNA sequences are compared to a nucleic acid sequenceencoding the subject DNAs or a fragment thereof having a size of atleast about 50 nucleotides. This includes allelic and splice variants ofthe subject genes. The present invention also encompasses nucleic acidsequences that hybridize to the subject splice variants under high,moderate or low stringency conditions e.g., as described infra.

Also, the present invention provides for primer pairs that result in theamplification of DNAs encoding the subject novel genes or a portionthereof in an mRNA library obtained from a desired cell source,typically human prostate cell or tissue sample. Typically, such primerswill be on the order of 12 to 50 nucleotides in length, and will beconstructed such that they provide for amplification of the entire ormost of the target gene.

Also, the invention embraces the antigens encoded by the subject DNAs orfragments thereof that bind to or elicits antibodies specific to thefull-length antigens. Typically, such fragments will be at least 10amino acids in length, more typically at least 25 amino acids in length.

As noted, the subject DNA fragments are expressed in a majority ofprostate tumor samples tested. The invention further contemplates theidentification of other cancers that express such genes and the usethereof to detect and treat such cancers. For example, the subject DNAfragments or variants thereof may be expressed on other cancers, e.g.,breast, ovary, pancreas, lung or prostate cancers. Essentially, thepresent invention embraces the detection of any cancer wherein theexpression of the subject novel genes or variants thereof correlate to acancer or an increased likelihood of cancer. To facilitate under-studyof the invention, the following definitions are provided.

“Isolated tumor antigen or tumor protein” refers to any protein that isnot in its normal cellular environment. This includes by way of examplecompositions comprising recombinant proteins encoded by the genesdisclosed infra, pharmaceutical compositions comprising such purifiedproteins, diagnostic compositions comprising such purified proteins, andisolated protein compositions comprising such proteins. In preferredembodiments, an isolated prostate tumor protein according to theinvention will comprise a substantially pure protein, in that it issubstantially free of other proteins, preferably that is at least 90%pure, that comprises the amino acid sequence contained herein or naturalhomologues or mutants having essentially the same sequence. A naturallyoccurring mutant might be found, for instance, in tumor cells expressinga gene encoding a mutated protein according to the invention.

“Native tumor antigen or tumor protein” refers to a protein that is anon-human primate homologue of the protein having the amino acidsequence contained infra.

“Isolated prostate tumor gene or nucleic acid sequence” refers to anucleic acid molecule that encodes a tumor antigen according to theinvention which is not in its normal human cellular environment, e.g.,is not comprised in the human or non-human primate chromosomal DNA. Thisincludes by way of example vectors that comprise a gene according to theinvention, a probe that comprises a gene according to the invention, anda nucleic acid sequence directly or indirectly attached to a detectablemoiety, e.g. a fluorescent or radioactive label, or a DNA fusion thatcomprises a nucleic acid molecule encoding a gene according to theinvention fused at its 5′ or 3′ end to a different DNA, e.g. a promoteror a DNA encoding a detectable marker or effector moiety. Also includedare natural homologues or mutants having substantially the samesequence. Naturally occurring homologies that are degenerate wouldencode the same protein including nucleotide differences that do notchange the corresponding amino acid sequence. Naturally occurringmutants might be found in tumor cells, wherein such nucleotidedifferences may result in a mutant tumor antigen. Naturally occurringhomologues containing conservative substitutions are also encompassed.

“Variant of prostate tumor antigen or tumor protein” refers to a proteinpossessing an amino acid sequence that possess at least 90% sequenceidentity, more preferably at least 91% sequence identity, even morepreferably at least 92% sequence identity, still more preferably atleast 93% sequence identity, still more preferably at least 94% sequenceidentity, even more preferably at least 95% sequence identity, stillmore preferably at least 96% sequence identity, even more preferably atleast 97% sequence identity, still more preferably at least 98% sequenceidentity, and most preferably at least 99% sequence identity, to thecorresponding native tumor antigen wherein sequence identity is asdefined infra. Preferably, this variant will possess at least onebiological property in common with the native protein.

“Variant of prostate tumor gene or nucleic acid molecule or sequence”refers to a nucleic acid sequence that possesses at least 90% sequenceidentity, more preferably at least 91%, more preferably at least 92%,even more preferably at least 93%, still more preferably at least 94%,even more preferably at least 95%, still more preferably at least 96%,even more preferably at least 97%, even more preferably at least 98%sequence identity, and most preferably at least 99% sequence identity,to the corresponding native human nucleic acid sequence, wherein“sequence identity” is as defined infra.

“Fragment of prostate antigen encoding nucleic acid molecule orsequence” refers to a nucleic acid sequence corresponding to a portionof the native human gene wherein said portion is at least about 50nucleotides in length, or 100, more preferably at least 150 nucleotidesin length.

“Antigenic fragments of prostate tumor antigen” refer to polypeptidescorresponding to a fragment of a prostate protein or a variant orhomologue thereof that when used itself or attached to an immunogeniccarrier elicits antibodies that specifically bind the protein. Typicallysuch antigenic fragments will be at least 8-15 amino acids in length,and may be much longer.

Sequence identity or percent identity is intended to mean the percentageof the same residues shared between two sequences, referenced to humanprotein A or protein B or gene A or gene B, when the two sequences arealigned using the Clustal method [Higgins et al, Cabios 8:189-191(1992)] of multiple sequence alignment in the Lasergene biocomputingsoftware (DNASTAR, INC, Madison, Wis.), or alignment programs availablefrom the Genetics Computer Group (GCG Wisconsin package, Accelrys, SanDiego, Calif.). In this method, multiple alignments are carried out in aprogressive manner, in which larger and larger alignment groups areassembled using similarity scores calculated from a series of pairwisealignments. Optimal sequence alignments are obtained by finding themaximum alignment score, which is the average of all scores between theseparate residues in the alignment, determined from a residue weighttable representing the probability of a given amino acid changeoccurring in two related proteins over a given evolutionary interval.Penalties for opening and lengthening gaps in the alignment contributeto the score. The default parameters used with this program are asfollows: gap penalty for multiple alignment=10; gap length penalty formultiple alignment=10; k-tuple value in pairwise alignment=1; gappenalty in pairwise alignment=3; window value in pairwise alignment=5;diagonals saved in pairwise alignment=5. The residue weight table usedfor the alignment program is PAM25O [Dayhoffet al., in Atlas of ProteinSequence and Structure, Dayhoff, Ed., NDRF, Washington, Vol. 5, suppl.3, p. 345, (1978)].

Percent conservation is calculated from the above alignment by addingthe percentage of identical residues to the percentage of positions atwhich the two residues represent a conservative substitution (defined ashaving a log odds value of greater than or equal to 0.3 in the PAM250residue weight table). Conservation is referenced to human Gene A orgene B when determining percent conservation with non-human Gene A orgene B, e.g. mgene A or gene B, when determining percent conservation.Conservative amino acid changes satisfying this requirement include:R-K; E-D, Y-F, L-M; V-I, Q-H.

Polypeptide Fragments

The invention provides polypeptide fragments of the disclosed proteins.Polypeptide fragments of the invention can comprise at least 8, morepreferably at least 25, still more preferably at least 50 amino acidresidues of the protein or an analogue thereof. More particularly suchfragment will comprise at least 75, 100, 125, 150, 175, 200, 225, 250,275 residues of the polypeptide encoded by the corresponding gene. Evenmore preferably, the protein fragment will comprise the majority of thenative protein, e.g. about 100 contiguous residues of the nativeprotein.

Biologically Active Variants

The invention also encompasses mutants of the novel prostate proteinsdisclosed infra which comprise an amino acid sequence that is at least80%, more preferably 90%, still more preferably 95-99% similar to thenative protein.

Guidance in determining which amino acid residues can be substituted,inserted, or deleted without abolishing biological or immunologicalactivity can be found using computer programs well known in the art,such as DNASTAR or software from the Genectics Computer Group (GCG).Preferably, amino acid changes in protein variants are conservativeamino acid changes, i.e., substitutions of similarly charged oruncharged amino acids. A conservative amino acid change involvessubstitution of one of a family of amino acids which are related intheir side chains. Naturally occurring amino acids are generally dividedinto four families: acidic (aspartate, glutamate), basic (lysine,arginine, histidine), non-polar (alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), and uncharged polar(glycine, asparagine, glutamine, cystine, serine, threonine, tyrosine)amino acids. Phenylalanine, tryptophan, and tyrosine are sometimesclassified jointly as aromatic amino acids.

A subset of mutants, called muteins, is a group of polypeptides in whichneutral amino acids, such as serines, are substituted for cysteineresidues which do not participate in disulfide bonds. These mutants maybe stable over a broader temperature range than native secretedproteins. See Mark et al., U.S. Pat. No. 4,959,314.

It is reasonable to expect that an isolated replacement of a leucinewith an isoleucine or valine, an aspartate with a glutamate, a threoninewith a serine, or a similar replacement of an amino acid with astructurally related amino acid will not have a major effect on thebiological properties of the resulting secreted protein or polypeptidevariant.

Protein variants include glycosylated forms, aggregative conjugates withother molecules, and covalent conjugates with unrelated chemicalmoieties. Also, protein variants also include allelic variants, speciesvariants, and muteins. Truncations or deletions of regions which do notaffect the differential expression of the gene are also variants.Covalent variants can be prepared by linking functionalities to groupswhich are found in the amino acid chain or at the N— or C-terminalresidue, as is known in the art.

It will be recognized in the art that some amino acid sequence of theprostate proteins of the invention can be varied without significanteffect on the structure or function of the protein. If such differencesin sequence are contemplated, it should be remembered that there arecritical areas on the protein which determine activity. In general, itis possible to replace residues that form the tertiary structure,provided that residues performing a similar function are used. In otherinstances, the type of residue may be completely unimportant if thealteration occurs at a non-critical region of the protein. Thereplacement of amino acids can also change the selectivity of binding tocell surface receptors. Ostade et al., Nature 361:266-268 (1993)describes certain mutations resulting in selective binding of TNF-alphato only one of the two known types of TNF receptors. Thus, thepolypeptides of the present invention may include one or more amino acidsubstitutions, deletions or additions, either from natural mutations orhuman manipulation.

The invention further includes variations of the prostate proteinsdisclosed infra which show comparable expression patterns or whichinclude antigenic regions. Such mutants include deletions, insertions,inversions, repeats, and site substitutions. Guidance concerning whichamino acid changes are likely to be phenotypically silent can be foundin Bowie, J. U., et al., “Deciphering the Message in Protein Sequences:Tolerance to Amino Acid Substitutions,” Science 247:1306-1310 (1990).

Of particular interest are substitutions of charged amino acids withanother charged amino acid and with neutral or negatively charged aminoacids. The latter results in proteins with reduced positive charge toimprove the characteristics of the disclosed protein. The prevention ofaggregation is highly desirable. Aggregation of proteins not onlyresults in a loss of activity but can also be problematic when preparingpharmaceutical formulations, because they can be imnmunogenic. (Pinckardet al., Clin. Exp. Immunol. 2:331-340 (1967); Robbins et al., Diabetes36:838-845 (1987); Cleland et al., Crit. Rev. Therapeutic Drug CarrierSystems 10:307-377 (1993)).

Amino acids in the polypeptides of the present invention that areessential for function can be identified by methods known in the art,such as site-directed mutagenesis or alanine-scanning mutagenesis(Cunningham and Wells, Science 244: 1081-1085 (1989)). The latterprocedure introduces single alanine mutations at every residue in themolecule. The resulting mutant molecules are then tested for biologicalactivity such as binding to a natural or synthetic binding partner.Sites that are critical for ligand-receptor binding can also bedetermined by structural analysis such as crystallization, nuclearmagnetic resonance or photoaffinity labeling (Smith et al., J Mol. Biol.224:899-904(1992) and de Vos et al. Science 255: 306-312 (1992)).

As indicated, changes are preferably of a minor nature, such asconservative amino acid substitutions that do not significantly affectthe folding or activity of the protein. Of course, the number of aminoacid substitutions a skilled artisan would make depends on many factors,including those described above. Generally speaking, the number ofsubstitutions for any given polypeptide will not be more than 50, 40,30, 25, 20, 15, 10, 5 or 3.

Fusion Proteins

Fusion proteins comprising proteins or polypeptide fragments of thesubject prostate tumor antigen can also be constructed. Fusion proteinsare useful for generating antibodies against amino acid sequences andfor use in various assay systems. For example, fusion proteins can beused to identify proteins which interact with a protein of the inventionor which interfere with its biological function. Physical methods, suchas protein afinnity chromatography, or library-based assays forprotein-protein interactions, such as the yeast two-hybrid or phagedisplay systems, can also be used for this purpose. Such methods arewell known in the art and can also be used as drug screens. Fusionproteins comprising a signal sequence and/or a transmembrane domain of aprotein according to the invention or a fragment thereof can be used totarget other protein domains to cellular locations in which the domainsare not normally found, such as bound to a cellular membrane or secretedextracellularly.

A fusion protein comprises two protein segments fused together by meansof a peptide bond. As noted, these fragments may range in size fromabout 8 amino acids up to the full length of the protein.

The second protein segment can be a full-length protein or a polypeptidefragment.

Proteins commonly used in fusion protein construction includeβ-galactosidase, β-glucuronidase, green fluorescent protein (GFP),autofluorescent proteins, including blue fluorescent protein (BFP),glutathione-S-transferase (GST), luciferase, horseradish peroxidase(HRP), and chloramphenicol acetyltransferase (CAT). Additionally,epitope tags can be used in fusion protein constructions, includinghistidine (His) tags, FLAG tags, influenza hemagglutinin (HA) tags, Myctags, VSV-G tags, and thioredoxin (Trx) tags. Other fusion constructionscan include maltose binding protein (MBP), S-tag, Lex a DNA bindingdomain (DBD) fusions, GALA DNA binding domain fusions, and herpessimplex virus (HSV) BP 16 protein fusions.

These fusions can be made, for example, by covalently linking twoprotein segments or by standard procedures in the art of molecularbiology. Recombinant DNA methods can be used to prepare fusion proteins,for example, by making a DNA construct which comprises a coding sequenceencoding a possible antigen according to the invention or a fragmentthereof in proper reading frame with a nucleotide encoding the secondprotein segment and expressing the DNA construct in a host cell, as isknown in the art. Many kits for constructing fusion proteins areavailable from companies that supply research labs with tools forexperiments, including, for example, Promega Corporation (Madison,Wis.), Stratagene (La Jolla, Calif.), Clontech (Mountain View, Calif.),Santa Cruz Biotechnology (Santa Cruz, Calif.), MBL InternationalCorporation (MIC; Watertown, Mass.), and Quantum Biotechnologies(Montreal, Canada; 1-888-DNA-KITS).

Proteins, fusion proteins, or polypeptides of the invention can beproduced by recombinant DNA methods. For production of recombinantproteins, fusion proteins, or polypeptides, a sequence encoding theprotein can be expressed in prokaryotic or eukaryotic host cells usingexpression systems known in the art. These expression systems includebacterial, yeast, insect, and mammalian cells.

The resulting expressed protein can then be purified from the culturemedium or from extracts of the cultured cells using purificationprocedures known in the art. For example, for proteins fully secretedinto the culture medium, cell-free medium can be diluted with sodiumacetate and contacted with a cation exchange resin, followed byhydrophobic interaction chromatography. Using this method, the desiredprotein or polypeptide is typically greater than 95% pure. Furtherpurification can be undertaken, using, for example, any of thetechniques listed above.

It may be necessary to modify a protein produced in yeast or bacteria,for example by phosphorylation or glycosylation of the appropriatesites, in order to obtain a functional protein. Such covalentattachments can be made using known chemical or enzymatic methods.

A protein or polypeptide of the invention can also be expressed incultured host cells in a form which will facilitate purification. Forexample, a protein or polypeptide can be expressed as a fusion proteincomprising, for example, maltose binding protein,glutathione-S-transferase, or thioredoxin, and purified using acommercially available kit. Kits for expression and purification of suchfusion proteins are available from companies such as New EnglandBioLabs, Pharmacia, and Invitrogen. Proteins, fusion proteins, orpolypeptides can also be tagged with an epitope, such as a “Flag”epitope (Kodak), and purified using an antibody which specifically bindsto that epitope.

The coding sequence of the protein variants identified through thesequences disclosed herein can also be used to construct transgenicanimals, such as mice, rats, guinea pigs, cows, goats, pigs, or sheep.Female transgenic animals can then produce proteins, polypeptides, orfusion proteins of the invention in their milk. Methods for constructingsuch animals are known and widely used in the art.

Alternatively, synthetic chemical methods, such as solid phase peptidesynthesis, can be used to synthesize a secreted protein or polypeptide.General means for the production of peptides, analogs or derivatives areoutlined in Chemistry and Biochemistry of Amino Acids, Peptides, andProteins—A Survey of Recent Developments, B. Weinstein, ed. (1983).Substitution of D-amino acids for the normal L-stereoisomer can becarried out to increase the half-life of the molecule.

Typically, homologous polynucleotide sequences can be confirmed byhybridization under stringent conditions, as is known in the art. Forexample, using the following wash conditions: 2×SSC (0.3 M NaCl, 0.03 Msodium citrate, pH 7.0), 0.1% SDS, room temperature twice, 30 minuteseach; then 2×SSC, 0.1% SDS, 50° C. once, 30 minutes; then 2×SSC, roomtemperature twice, 10 minutes each, homologous sequences can beidentified which contain at most about 25-30% basepair mismatches. Morepreferably, homologous nucleic acid strands contain 15-25% basepairmismatches, even more preferably 5-15% basepair mismatches.

The invention also provides polynucleotide probes which can be used todetect complementary nucleotide sequences, for example, in hybridizationprotocols such as Northern or Southern blotting or in situhybridizations. Polynucleotide probes of the invention comprise at least12, 13, 14, 15, 16, 17, 18, 19, 20, 30, or 40 or more contiguousnucleotides of the nucleic acid sequences provided herein.Polynucleotide probes of the invention can comprise a detectable label,such as a radioisotopic, fluorescent, enzymatic, or chemiluminescentlabel.

Isolated genes corresponding to the cDNA sequences disclosed herein arealso provided. Standard molecular biology methods can be used to isolatethe corresponding genes using the cDNA sequences provided herein. Thesemethods include preparation of probes or primers from the nucleotidesequence disclosed herein for use in identifying or amplifying the genesfrom mammalian, including human, genomic libraries or other sources ofhuman genomic DNA.

Polynucleotide molecules of the invention can also be used as primers toobtain additional copies of the polynucleotides, using polynucleotideamplification methods. Polynucleotide molecules can be propagated invectors and cell lines using techniques well known in the art.Polynucleotide molecules can be on linear or circular molecules. Theycan be on autonomously replicating molecules or on molecules withoutreplication sequences. They can be regulated by their own or by otherregulatory sequences, as is known in the art.

Polynucleotide Constructs

Polynucleotide molecules comprising the coding sequences of the genevariants identified through the sequences disclosed herein can be usedin a polynucleotide construct, such as a DNA or RNA construct.Polynucleotide molecules of the invention can be used, for example, inan expression construct to express all or a portion of a protein,variant, fusion protein, or single-chain antibody in a host cell. Anexpression construct comprises a promoter which is functional in achosen host cell. The skilled artisan can readily select an appropriatepromoter from the large number of cell type-specific promoters known andused in the art. The expression construct can also contain atranscription terminator which is functional in the host cell. Theexpression construct comprises a polynucleotide segment which encodesall or a portion of the desired protein. The polynucleotide segment islocated downstream from the promoter. Transcription of thepolynucleotide segment initiates at the promoter. The expressionconstruct can be linear or circular and can contain sequences, ifdesired, for autonomous replication.

Also included are polynucleotide molecules comprising the promoter andUTR sequences of the subject novel genes, operably linked to theassociated protein coding sequence and/or other sequences encoding adetectable or selectable marker. Such promoter and/or UTR-basedconstructs are useful for studying the transcriptional and translationalregulation of protein expression, and for identifying activating and/orinhibitory regulatory proteins.

Host Cells

An expression construct can be introduced into a host cell. The hostcell comprising the expression construct can be any suitable prokaryoticor eukaryotic cell. Expression systems in bacteria include thosedescribed in Chang et al., Nature 275:615 (1978); Goeddel et al., Nature281: 544 (1979); Goeddel et al., Nucleic Acids Res. 8:4057 (1980); EP36,776; U.S. Pat. No. 4,551,433; deBoer et al., Proc. Natl. Acad Sci.USA 80: 21-25 (1983); and Siebenlist et al., Cell 20: 269 (1980).

Expression systems in yeast include those described in Hinnnen et al.,Proc. Natl. Acad. Sci. USA 75: 1929 (1978); Ito et al., J Bacteriol 153:163 (1983); Kurtz et al., Mol. Cell. Biol. 6: 142 (1986); Kunze et al.,J Basic Microbiol. 25: 141 (1985); Gleeson et al., J. Gen. Microbiol.132: 3459 (1986), Roggenkamp et al., Mol. Gen. Genet. 202: 302 (1986));Das et al., J Bacteriol. 158: 1165 (1984); De Louvencourt et al., JBacteriol. 154:737 (1983), Van den Berg et al., Bio/Technology 8: 135(1990); Kunze et al., J. Basic Microbiol. 25: 141 (1985); Cregg et al.,Mol. Cell. Biol. 5: 3376 (1985); U.S. Pat. No. 4,837,148; U.S. Pat. No.4,929,555; Beach and Nurse, Nature 300: 706 (1981); Davidow et al.,Curr. Genet. 10: 380(1985); Gaillardin et al., Curr. Genet. 10: 49(1985); Ballance et al., Biochem. Biophys. Res. Commun. 112: 284-289(1983); Tilburn et al., Gene 26: 205-22 (1983); Yelton et al., Proc.Natl. Acad, Sci. USA 81: 1470-1474 (1984); Kelly and Hynes, EMBO J. 4:475479 (1985); EP 244,234; and WO 91/00357.

Expression of heterologous genes in insects can be accomplished asdescribed in U.S. Pat. No. 4,745,051; Friesen et al. (1986) “TheRegulation of Baculovirus Gene Expression” in: THE MOLECULAR BIOLOGY OFBACULOVIRUSES (W. Doerfler, ed.); EP 127,839; EP 155,476; Vlak et al.,J. Gen. Virol. 69: 765-776 (1988); Miller et al., Ann. Rev. Microbiol.42: 177 (1988); Carbonell et al., Gene 73: 409 (1988); Maeda et al.,Nature 315: 592-594 (1985); Lebacq-Verheyden et al., Mol. Cell Biol. 8:3129 (1988); Smith et al., Proc. Natl. Acad. Sci. USA 82: 8404 (1985);Miyajima et al., Gene 58: 273 (1987); and Martin et al., DNA 7:99(1988). Numerous baculoviral strains and variants and correspondingpermissive insect host cells from hosts are described in Luckow et al.,Bio/Technology (1988) 6: 47-55, Miller et al., in GENETIC ENGINEERING(Setlow, J. K. et al. eds.), Vol. 8, pp. 277-279 (Plenum Publishing,1986); and Maeda et al., Nature, 315: 592-594 (1985).

Mammalian expression can be accomplished as described in Dijkema et al.,EMBO J. 4: 761(1985); Gormanetal., Proc. Natl. Acad. Sci. USA 79: 6777(1982b); Boshart et al., Cell 41: 521 (1985); and U.S. Pat. No.4,399,216. Other features of mammalian expression can be facilitated asdescribed in Ham and Wallace, Meth Enz. 58: 44 (1979);

Expression constructs can be introduced into host cells using anytechnique known in the art. These techniques includetransferrin-polycation-mediated DNA transfer, transfection with naked orencapsulated nucleic acids, liposome-mediated cellular fusion,intracellular transportation of DNA-coated latex beads, protoplastfusion, viral infection, electroporation, “gene gun,” and calciumphosphate-mediated transfection.

The invention can also include hybrid and modified forms thereofincluding fusion proteins, fragments and hybrid and modified forms inwhich certain amino acids have been deleted or replaced, modificationssuch as where one or more amino acids have been changed to a modifiedamino acid or unusual amino acid.

Also included within the meaning of substantially homologous is anyhuman or non-human primate protein which may be isolated by virtue ofcross-reactivity with antibodies to proteins encoded by a gene describedherein or whose encoding nucleotide sequences including genomic DNA,mRNA or cDNA may be isolated through hybridization with thecomplementary sequence of genomic or subgenomic nucleotide sequences orcDNA of a gene herein or fragments thereof It will also be appreciatedby one skilled in the art that degenerate DNA sequences can encode atumor protein according to the invention and these are also intended tobe included within the present invention as are allelic variants of thesubject genes.

Preferred is a prostate protein according to the invention prepared byrecombinant DNA technology. By “pure form” or “purified form” or“substantially purified form” it is meant that a protein composition issubstantially free of other proteins which are not the desired protein.

The present invention also includes therapeutic or pharmaceuticalcompositions comprising a protein according to the invention in aneffective amount for treating patients with disease, and a methodcomprising administering a therapeutically effective amount of theprotein. These compositions and methods are useful for treating cancersassociated with the subject proteins, e.g. prostate cancer. One skilledin the art can readily use a variety of assays known in the art todetermine whether the protein would be useful in promoting survival orfunctioning in a particular cell type.

Anti-Prostate Antigen Antibodies

As noted, the invention includes the preparation and use ofanti-prostate antigen antibodies and fragments for use as diagnosticsand therapeutics. These antibodies may be polyclonal or monoclonal.Polyclonal antibodies can be prepared by immunizing rabbits or otheranimals by injecting antigen followed by subsequent boosts atappropriate intervals. The animals are bled and sera assayed againstpurified protein usually by ELISA or by bioassay based upon the abilityto block the action of the corresponding gene. When using avian species,e.g., chicken, turkey and the like, the antibody can be isolated fromthe yolk of the egg. Monoclonal antibodies can be prepared after themethod of Milstein and Kohler by fusing splenocytes from immunized micewith continuously replicating tumor cells such as myeloma or lymphomacells. [Milstein and Kohler, Nature 256:495-497 (1975); Gulfre andMilstein, Methods in Enzymology: Immunochemical Techniques 73:1-46,Langone and Banatis eds., Academic Press, (1981) which are incorporatedby reference]. The hybridoma cells so formed are then cloned by limitingdilution methods and supernates assayed for antibody production byELISA, RIA or bioassay.

The unique ability of antibodies to recognize and specifically bind totarget proteins provides an approach for treating an overexpression ofthe protein. Thus, another aspect of the present invention provides fora method for preventing or treating diseases involving overexpression ofthe protein by treatment of a patient with specific antibodies to theprotein.

Specific antibodies, either polyclonal or monoclonal, to the protein canbe produced by any suitable method known in the art as discussed above.For example, by recombinant methods, preferably in eukaryotic cellsmurine or human monoclonal antibodies can be produced by hybridomatechnology or, alternatively, the protein, or an immunologically activefragment thereof, or an anti-idiotypic antibody, or fragment thereof canbe administered to an animal to elicit the production of antibodiescapable of recognizing and binding to the protein. Such antibodies canbe from any class of antibodies including, but not limited to IgG, IgA,1 gM, IgD, and IgE or in the case of avian species, IgY and from anysubclass of antibodies.

The availability of isolated protein allows for the identification ofsmall molecules and low molecular weight compounds that inhibit thebinding of protein to binding partners, through routine application ofhigh-throughput screening methods (HTS). HTS methods generally refer totechnologies that permit the rapid assaying of lead compounds fortherapeutic potential. HTS techniques employ robotic handling of testmaterials, detection of positive signals, and interpretation of data.Lead compounds may be identified via the incorporation of radioactivityor through optical assays that rely on absorbance, fluorescence orluminescence as read-outs. [Gonzalez, J. E. et al., Curr. Opin. Biotech.9:624-63 1 (1998)].

Model systems are available that can be adapted for use in highthroughput screening for compounds that inhibit the interaction ofprotein with its ligand, for example by competing with protein forligand binding. Sarubbi et al., Anal. Biochem. 237:70-75(1996) describecell-free, non-isotopic assays for discovering molecules that competewith natural ligands for binding to the active site of IL-1 receptor.Martens, C. et al., Anal. Biochem. 273:20-31 (1999) describe a genericparticle-based nonradioactive method in which a labeled ligand binds toits receptor immobilized on a particle; label on the particle decreasesin the presence of a molecule that competes with the labeled ligand forreceptor binding.

Antibody Preparation

(i) Starting Materials and Methods

Immunoglobulins (Ig) and certain variants thereof are known and manyhave been prepared in recombinant cell culture. For example, see U.S.Pat. No. 4,745,055; EP 256,654; EP 120,694; EP 125,023; EP 255,694; EP266,663; WO 30 88/03559; Faulkneret al., Nature, 298: 286 (1982);Morrison, J. Immun., 123: 793 (1979); Koehler et al., Proc. Natl. Acad.Sci. USA, 77: 2197 (1980); Raso et al., Cancer Res., 41: 2073 (1981);Morrison et al., Ann. Rev. Immunol., 2: 239 (1984); Morrison, Science,229: 1202 (1985); and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851 (1984). Reassorted immunoglobulin chains are also known. See, forexample, U.S. Pat. No. 4,444,878; WO 88/03565; and EP 68,763 andreferences cited therein. The immunoglobulin moiety in the chimeras ofthe present invention may be obtained from IgG-1, IgG-2, IgG-3, or IgG-4subtypes, IgA, IgE, IgD, or IgM, but preferably from IgG-1 or IgG-3.

(ii) Polyclonal Antibodies

Polyclonal antibodies to the subject prostate antigens are generallyraised in animals by multiple subcutaneous (sc) or intraperitoneal (ip)injections of the antigen and an adjuvant. It maybe useful to conjugatethe antigen or a fragment containing the target amino acid sequence to aprotein that is immunogenic in the species to be immunized, e.g.,keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, orsoybean trypsin inhibitor using a bifunctional or derivatizing agent,for example, maleimidobenzoyl sulfosuccinimide ester (conjugationthrough cysteine residues), N-hydroxysuccinimide (through lysineresidues), glutaraldehyde or succinic anhydride.

Animals are immunized against the polypeptide or fragment, immunogenicconjugates, or derivatives by combining about 1 mg or 1 μg of thepeptide or conjugate (for rabbits or mice, respectively) with 3 volumesof Freund's complete adjuvant and injecting the solution intradermallyat multiple sites. One month later the animals are boosted with ⅕ to1/10 the original amount of peptide or conjugate in Freund's completeadjuvant by subcutaneous injection at multiple sites. Seven to 14 dayslater the animals are bled and the serum is assayed for antibody titerto the antigen or a fragment thereof. Animals are boosted until thetiter plateaus. Preferably, the animal is boosted with the conjugate ofthe same polypeptide or fragment thereof, but conjugated to a differentprotein and/or through a different cross-linking reagent. Conjugatesalso can be made in recombinant cell culture as protein fusions. Also,aggregating agents such as alum are suitably used to enhance the immuneresponse.

(iii) Monoclonal Antibodies

Monoclonal antibodies are obtained from a population of substantiallyhomogeneous antibodies, i.e., the individual antibodies comprising thepopulation are identical except for possible naturally occurringmutations that may be present in minor amounts. Thus, the modifier“monoclonal” indicates the character of the antibody as not being amixture of discrete antibodies.

For example, monoclonal antibodies using for practicing this inventionmay be made using the hybridoma method first described by Kohler andMilstein, Nature, 256: 495 (1975), or may be made by recombinant DNAmethods (Cabilly et al., supra).

In the hybridoma method, a mouse or other appropriate host animal, suchas a hamster, is immunized as hereinabove described to elicitlymphocytes that produce or are capable of producing antibodies thatwill specifically bind to the antigen or fragment thereof used forimmunization. Alternatively, lymphocytes maybe immunized in vitro.Lymphocytes then are fused with myeloma cells using a suitable fusingagent, such as polyethylene glycol, to form a hybridoma cell (Goding,Monoclonal Antibodies: Principles and Practice, pp. 59-103 [AcademicPress, 1986]).

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Preferred myeloma cells are those that fuse efficiently, support stablehigh-level production of antibody by the selected antibody-producingcells, and are sensitive to a medium such as HAT medium. Among these,preferred myeloma cell lines are murine myeloma lines, such as thosederived from MOPC-21 and MPC-11 mouse tumors available from the SalkInstitute Cell Distribution Center, San Diego, Calif. USA, and SP-2cells available from the American Type Culture Collection, Rockville,Md. USA.

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against the prostateantigen. Preferably, the binding specificity of monoclonal antibodiesproduced by hybridoma cells is determined by immunoprecipitation or byan in vitro binding assay, such as radioimmunoassay (RIA) orenzyme-linked immunoabsorbent assay (ELISA).

The binding affinity of the monoclonal antibody can, for example, bedetermined by the Scatchard analysis of Munson and Pollard, Anal.Biochem., 107: 220 (1980).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, supra). Suitable culture media for this purpose include, forexample, D-MEM or RPMI-1640 medium. In addition, the hybridoma cellsmaybe grown in vivo as ascites tumors in an animal.

The monoclonal antibodies secreted by the subdlones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxyapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

DNA encoding the monoclonal antibodies of the invention is readilyisolated and sequenced using conventional procedures (e.g., by usingoligonucleotide probes that are capable of binding specifically to genesencoding the heavy and light chains of murine antibodies). The hybridomacells of the invention serve as a preferred source of such DNA. Onceisolated, the DNA maybe placed into expression vectors, which are thentransfected into host cells such as E. Coli cells, simian COS cells,Chinese hamster ovary (CHO) cells, or myeloma cells that do nototherwise produce immunoglobulin protein, to obtain the synthesis ofmonoclonal antibodies in the recombinant host cells. Review articles onrecombinant expression in bacteria of DNA encoding the antibody includeSkerra et al., Curr. Opinion in Immunol., 5: 256-262 (1993) andPluckthun, Immunol. Revs., 130: 151-188 (1992).

The DNA also may be modified, for example, by substituting the codingsequence for human heavy- and light-chain constant domains in place ofthe homologous murine sequences (Morrison, et al., Proc. Natl. Acad.Sci. USA, 81: 6851 [1984]), or by covalently joining to theimmunoglobulin coding sequence all or part of the coding sequence for anon-immunoglobulin polypeptide. In that manner, “chimeric” or “hybrid”antibodies are prepared that have the binding specificity of ananti-prostate antigen monoclonal antibody herein.

Typically such non-immunoglobulin polypeptides are substituted for theconstant domains of an antibody of the invention, or they aresubstituted for the variable domains of one antigen-combining site of anantibody of the invention to create a chimeric bivalent antibodycomprising one antigen-combining site having specificity for prostateantigen according to the invention and another antigen-combining sitehaving specificity for a different antigen.

Chimeric or hybrid antibodies also may be prepared in vitro using knownmethods in synthetic protein chemistry, including those involvingcrosslinking agents. For example, immunotoxins may be constructed usinga disulfide-exchange reaction or by forming a thioether bond. Examplesof suitable reagents for this purpose include iminothiolate andmethyl-4-mercaptobutyrimidate.

(iv) Humanized Antibodies

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers(Jones et al., Nature 321, 522-525 [1986]; Riechmann et al., Nature332,323-327 [1988]; Verhoeyen et al., Science 239,1534-1536 [1988]), bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (Cabilly et al., supra), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable domain of a rodent antibody is screened against theentire library of known human variable-domain sequences. The humansequence which is closest to that of the rodent is then accepted as thehuman framework (FR) for the humanized antibody (Sims et al., J.Immunol., 151: 2296 [1993]; Chothia and Lesk, J. Mol. Biol., 196: 901[1987]). Another method uses a particular framework derived from theconsensus sequence of all human antibodies of a particular subgroup oflight or heavy chains. The same framework may be used for severaldifferent humanized antibodies (Carter et al., Proc. Natl. Acad. Sci.USA, 89: 4285 [1992]; Presta et al., J. Immnol., 151: 2623 [1993]).

It is further important that antibodies be humanized with retention ofhigh affinity for the antigen and other favorable biological properties.To achieve this goal, according to a preferred method, humanizedantibodies are prepared by a process of analysis of the parentalsequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the consensus and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the CDR residues aredirectly and most substantially involved in influencing antigen binding.

(v) Human Antibodies

Human monoclonal antibodies can be made by the hybridoma method. Humanmyeloma and mouse-human heteromyeloma cell lines for the production ofhuman monoclonal antibodies have been described, for example, by Kozbor,J. Immunol. 133, 3001 (1984); Brodeur, et al., Monoclonal AntibodyProduction Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc.,New York, 1987); and Boerner et al., J. Immunol., 147: 86-95 (1991).

It is now possible to produce transgenic animals (e.g., mice) that arecapable, upon immunization, of producing a full repertoire of humanantibodies in the absence of endogenous immunoglobulin production. Forexample, it has been described that the homozygous deletion of theantibody heavy-chain joining region (J_(H)) gene in chimeric andgerm-line mutant mice results in complete inhibition of endogenousantibody production. Transfer of the human germ-line immunoglobulin genearray in such germ-line mutant mice will result in the production ofhuman antibodies upon antigen challenge. See, e.g., Jakobovits et al.,Proc. Natl. Acad. Sci. USA, 90: 2551 (1993); Jakobovits et al., Nature,362: 255-258 (1993); Bruggermann et al., Year in Immuno., 7: 33 (1993).

Alternatively, the phage display technology (McCafferty et al., Nature,348: 552-553 [1990]) can be used to produce human antibodies andantibody fragments in vitro, from immunoglobulin variable (V) domaingene repertoires from non-immunized donors. According to this technique,antibody V domain genes are cloned in-frame into either a major or minorcoat protein gene of a filamentous bacteriophage, such as M13 or fd, anddisplayed as functional antibody fragments on the surface of the phageparticle. Because the filamentous particle contains a single-strandedDNA copy of the phage genome, selections based on the functionalproperties of the antibody also result in selection of the gene encodingthe antibody exhibiting those properties. Thus, the phage mimics some ofthe properties of the B-cell. Phage display can be performed in avariety of formats; for their review see, e.g., Johnson and Chiswell,Curr. Op. Struct. Biol., 3: 564-571 (1993). Several sources of V-genesegments can be used for phage display. Clackson et al., Nature, 352:624-628 (1991) isolated a diverse array of anti-oxazolone antibodiesfrom a small random combinatorial library of V genes derived from thespleens of immunized mice. A repertoire of V genes from non-immunizedhuman donors can be constructed and antibodies to a diverse array ofantigens (including self-antigens) can be isolated essentially followingthe techniques described by Marks et al., J. Mol. Biol., 222: 581-597(1991), or Griffith et al., EMBO J., 12: 725-734 (1993).

In a natural immune response, antibody genes accumulate mutations at ahigh rate (somatic hypermutation). Some of the changes introduced willconfer higher affinity, and B cells displaying high-affinity surfaceimmunoglobulin are preferentially replicated and differentiated duringsubsequent antigen challenge. This natural process can be mimicked byemploying the technique known as “chain shuffling” (Marks et al.,Bio/Technology, 10: 779-783 [1992]). In this method, the affinity of“primary” human antibodies obtained by phage display can be improved bysequentially replacing the heavy and light chain V region genes withrepertoires of naturally occurring variants (repertoires) of V domaingenes obtained from non-immunized donors. This technique allows theproduction of antibodies and antibody fragments with affinities in thenM range. A strategy for making very large phage antibody repertoireshas been described by Waterhouse et al., Nucl. Acids Res., 21: 2265-2266(1993).

Gene shuffling can also be used to derive human antibodies from rodentantibodies, where the human antibody has similar affinities andspecificities to the starting rodent antibody. According to this method,which is also referred to as “epitope imprinting”, the heavy or lightchain V domain gene of rodent antibodies obtained by phage displaytechnique is replaced with a repertoire of human V domain genes,creating rodent-human chimeras. Selection on antigen results inisolation of human variable capable of restoring a functionalantigen-binding site, i.e., the epitope governs (imprints) the choice ofpartner. When the process is repeated in order to replace the remainingrodent V domain, a human antibody is obtained (see PCT WO 93/06213,published Apr. 1, 1993). Unlike traditional humanization of rodentantibodies by CDR grafting, this technique provides completely humanantibodies, which have no framework or CDR residues of rodent origin.

(vi) Bispecific Antibodies

Bispecific antibodies are monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens. In the present case, one of the binding specificities will beto a prostate antigen according to the invention. Methods for makingbispecific antibodies are known in the art.

Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy chain-light chainpairs, where the two heavy chains have different specificities (Milsteinand Cuello, Nature, 305: 537-539 [1983]). Because of the randomassortment of immunoglobulin heavy and light chains, these hybridomas(quadromas) produce a potential mixture of 10 different antibodymolecules, of which only one has the correct bispecific structure. Thepurification of the correct molecule, which is usually done by affinitychromatography steps, is rather cumbersome, and the product yields arelow. Similar procedures are disclosed in WO 93/08829 published May 13,1993, and in Traunecker et al., EMBO J., 10: 3655-3659 (1991).

According to a different and more preferred approach, antibody-variabledomains with the desired binding specificities(antibody-antigencombining sites) are fused to immunoglobulinconstant-domain sequences. The fusion preferably is with animmunoglobulin heavy-chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. It is preferred to have the firstheavy-chain constant region (CH1), containing the site necessary forlight-chain binding, present in at least one of the fusions. DNAsencoding the immunoglobulin heavy chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transfected into a suitable host organism. Thisprovides for great flexibility in adjusting the mutual proportions ofthe three polypeptide fragments in embodiments when unequal ratios ofthe three polypeptide chains used in the construction provide theoptimum yields. It is, however, possible to insert the coding sequencesfor two or all three polypeptide chains in one expression vector whenthe production of at least two polypeptide chains in equal ratiosresults in high yields or when the ratios are of no particularsignificance. In a preferred embodiment of this approach, the bispecificantibodies are composed of a hybrid immunoglobulin heavy chain with afirst binding specificity in one arm, and a hybrid immunoglobulin heavychain-light chain pair (providing a second binding specificity) in theother arm. It was found that this asymmetric structure facilitates theseparation of the desired bispecific compound from unwantedimmunoglobulin chain combinations, as the presence of an immunoglobulinlight chain in only one half of the bispecific molecule provides for afacile way of separation.

For further details of generating bispecific antibodies, see, forexample, Suresh et al., Methods in Enzymology, 121: 210 (1986).

(vii) Heteroconjuqate Antibodies

Heteroconjugate antibodies are also within the scope of the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune system cells to unwanted cells (U.S. Pat. No. 4,676,980),and for treatment of HIV infection (WO 91/00360; WO 92/00373; and EP03089). Heteroconjugate antibodies may be made using any convenientcross-linking methods. Suitable cross-linking agents are well known inthe art, and are disclosed in U.S. Pat. No. 4,676,980, along with anumber of cross-linking techniques.

The polynucleotides and polypeptides of the present invention maybeutilized in gene delivery vehicles. The gene delivery vehicle may be ofviral or non-viral origin (see generally, Jolly, Cancer Gene Therapy1:51-64 (1994); Kimura, Human Gene Therapy 5:845-852 (1994); Connelly,Human Gene Therapy 1:185-193 (1995); and Kaplitt, Nature Genetics6:148-153 (1994)). Gene therapy vehicles for delivery of constructsincluding a coding sequence of a therapeutic according to the inventioncan be administered either locally or systemically. These constructs canutilize viral or non-viral vector approaches. Expression of such codingsequences can be induced using endogenous mammalian or heterologouspromoters. Expression of the coding sequence can be either constitutiveor regulated. Preferred vehicles for gene therapy include retroviral andadeno-viral vectors.

Representative examples of adenoviral vectors include those described byBerkner, Biotechniques 6:616-627 (Biotechniques); Rosenfeld et al.,Science 252:431-434 (1991); WO 93/19191; Kolls et al., P.N.A.S.215-219(1994); Kass-Bisleret al., P.N.A.S. 90:11498-11502 (1993); Guzmanet al., Circulation 88: 2838-2848 (1993); Guzman et al., Cir. Res.73:1202-1207 (1993); Zabner et al., Cell 75: 207-216 (1993); Li et al.,Hum. Gene Other. 4: 403-409 (1993); Cailaud et al., Eur. J. Neurosci. 5:1287-1291 (1993); Vincent et al., Nat. Genet. 5: 130-134 (1993); Jaffeet al., Nat. Genet. 1: 372-378 (1992); and Levrero et al., Gene 101:195-202 (1992). Exemplary adenoviral gene therapy vectors employable inthis invention also include those described in WO 94/12649, WO 93/03769;WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655. Administration ofDNA linked to kill adenovirus as described in Curiel, Hum. Gene Other.3: 147-154 (1992) maybe employed.

Other gene delivery vehicles and methods may be employed; includingpolycationic condensed DNA linked or unlinked to kill adenovirus alone,for example Curiel, Hum. Gene Other. 3: 147-154 (1992); ligand-linkedDNA, for example see Wu, J. Biol. Chem. 264:16985-16987 (1989);eukaryotic cell delivery vehicles cells, for example see U.S. Ser. No.08/240,030, filed May 9, 1994, and U.S. Ser. No. 08/404,796; depositionof photopolymerized hydrogel materials; hand-held gene transfer particlegun, as described in U.S. Pat. No. 5,149,655; ionizing radiation asdescribed in U.S. Pat. No. 5,206,152 and in WO 92/11033; nucleic chargeneutralization or fusion with cell membranes. Additional approaches aredescribed in Philip, Mol. Cell Biol. 14:2411-2418 (1994), and inWoffendin, Proc. Natl. Acad. Sci. 91:1581-1585 (1994).

Naked DNA may also be employed. Exemplary naked DNA introduction methodsare described in WO 90/11092 and U.S. Pat. No. 5,580,859. Uptakeefficiency may be improved using biodegradable latex beads. DNA coatedlatex beads are efficiently transported into cells after endocytosisinitiation by the beads. The method may be improved further by treatmentof the beads to increase hydrophobicity and thereby facilitatedisruption of the endosome and release of the DNA into the cytoplasm.Liposomes that can act as gene delivery vehicles are described in U.S.Pat. No. 5,422,120, PCT Patent Publication Nos. WO 95/13796, WO94/23697, and WO 91/14445, and EP No. 0 524 968.

Further non-viral delivery suitable for use includes mechanical deliverysystems such as the approach described in Woffendin et al., Proc. Natl.Acad. Sci. USA 91(24): 11581-11585 (1994). Moreover, the coding sequenceand the product of expression of such can be delivered throughdeposition of photopolymerized hydrogel materials. Other conventionalmethods for gene delivery that can be used for delivery of the codingsequence include, for example, use of hand-held gene transfer particlegun, as described in U.S. Pat. No. 5,149,655; use of ionizing radiationfor activating transferred gene, as described in U.S. Pat. No. 5,206,152and PCT Patent Publication No. WO 92/11033.

The subject antibodies or antibody fragments maybe conjugated directlyor indirectly to effective moieties, e.g., radionuclides, toxins,chemotherapeutic agents, prodrugs, cytoslatic agents, enzymes and thelike. In a preferred embodiment the antibody or fragment will beattached to a therapeutic or diagnostic radiolabel directly or by use ofa chelating agent. Examples of suitable radiolabels are well known andinclude ⁹⁰Y, ¹²⁵I, ¹³¹I, ¹¹¹I, ¹⁰⁵Rh, ¹⁵³Sm, ⁶⁷Cu, ⁶⁷Ga, ¹⁶⁶Ho, ¹⁷⁷Lu,¹⁸⁶Re and ¹⁸⁸Re.

Examples of suitable drugs that my be coupled to antibodies includemethotrexate, adriamycine and lymphokines such as interferons,interleukins and the like. Suitable toxins which may be coupled includericin, cholera and diptheria toxin.

In a preferred embodiment, the subject antibodies will be attached to atherapeutic radiolabel and used for radioimmunotherapy.

Anti-Sense Oligonucleotides

In certain circumstances, it maybe desirable to modulate or decrease theamount of the protein expressed by a prostate cell. Thus, in anotheraspect of the present invention, anti-sense oligonucleotides can be madeand a method utilized for diminishing the level of expression a prostateantigen according to the invention by a cell comprising administeringone or more anti-sense oligonucleotides. By anti-sense oligonucleotidesreference is made to oligonucleotides that have a nucleotide sequencethat interacts through base pairing with a specific complementarynucleic acid sequence involved in the expression of the target such thatthe expression of the gene is reduced. Preferably, the specific nucleicacid sequence involved in the expression of the gene is a genomic DNAmolecule or mRNA molecule that encodes the gene. This genomic DNAmolecule can comprise regulatory regions of the gene, or the codingsequence for the mature gene.

The term complementary to a nucleotide sequence in the context ofantisense oligonucleotides and methods therefor means sufficientlycomplementary to such a sequence as to allow hybridization to thatsequence in a cell, i.e., under physiological conditions. Antisenseoligonucleotides preferably comprise a sequence containing from about 8to about 100 nucleotides and more preferably the antisenseoligonucleotides comprise from about 15 to about 30 nucleotides.Antisense oligonucleotides can also contain a variety of modificationsthat confer resistance to nucleolytic degradation such as, for example,modified internucleoside linages [Uhlmann and Peyman, Chemical Reviews90:543-548 (1990); Schneider and Banner, Tetrahedron Lett. 31:335,(1990) which are incorporated by reference], modified nucleic acid basesas disclosed in U.S. Pat. No. 5,958,773 and patents disclosed therein,and/or sugars and the like.

Any modifications or variations of the antisense molecule which areknown in the art to be broadly applicable to antisense technology areincluded within the scope of the invention. Such modifications includepreparation of phosphorus-containing linkages as disclosed in U.S. Pat.Nos. 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799;5,587,361,5,625,050 and 5,958,773.

The antisense compounds of the invention can include modified bases. Theantisense oligonucleotides of the invention can also be modified bychemically linking the oligonucleotide to one or more moieties orconjugates to enhance the activity, cellular distribution, or cellularuptake of the antisense oligonucleotide. Such moieties or conjugatesinclude lipids such as cholesterol, cholic acid, thioether, aliphaticchains, phospholipids, polyamines, polyethylene glycol (PEG), palmitylmoieties, and others as disclosed in, for example, U.S. Pat. Nos.5,514,758, 5,565,552, 5,567,810, 5,574,142, 5,585,481, 5,587,371,5,597,696 and 5,958,773.

Chimeric antisense oligonucleotides are also within the scope of theinvention, and can be prepared from the present inventiveoligonucleotides using the methods described in, for example, U.S. Pat.Nos. 5,013,830, 5,149,797, 5,403,711, 5,491,133, 5,565,350, 5,652,355,5,700,922 and 5,958,773.

In the antisense art a certain degree of routine experimentation isrequired to select optimal antisense molecules for particular targets.To be effective, the antisense molecule preferably is targeted to anaccessible, or exposed, portion of the target RNA molecule. Although insome cases information is available about the structure of target mRNAmolecules, the current approach to inhibition using antisense is viaexperimentation. mRNA levels in the cell can be measured routinely intreated and control cells by reverse transcription of the mRNA andassaying the cDNA levels. The biological effect can be determinedroutinely by measuring cell growth or viability as is known in the art.

Measuring the specificity of antisense activity by assaying andanalyzing cDNA levels is an art-recognized method of validatingantisense results. It has been suggested that RNA from treated andcontrol cells should be reverse-transcribed and the resulting cDNApopulations analyzed. (Branch, A. D., T.I.B.S. 23:45-50 (1998)].

The therapeutic or pharmaceutical compositions of the present inventioncan be administered by any suitable route known in the art including forexample intravenous, subcutaneous, intramuscular, transdermal,intrathecal or intracerebral. Administration can be either rapid as byinjection or over a period of time as by slow infusion or administrationof slow release formulation.

Additionally, the subject prostate tumor proteins can also be linked orconjugated with agents that provide desirable pharmaceutical orpharmacodynamic properties. For example, the protein can be coupled toany substance known in the art to promote penetration or transportacross the blood-brain barrier such as an antibody to the transferrinreceptor, and administered by intravenous injection (see, for example,Friden et al., Science 259:373-377 (1993) which is incorporated byreference). Furthermore, the subject protein A or protein B can bestably linked to a polymer such as polyethylene glycol to obtaindesirable properties of solubility, stability, half-life and otherpharmaceutically advantageous properties. [See, for example, Davis etal.,Enzyme Eng. 4:169-73 (1978); Buruham,Am. J. Hosp. Pharm. 51:210-218(1994) which are incorporated by reference].

The compositions are usually employed in the form of pharmaceuticalpreparations. Such preparations are made in a manner well known in thepharmaceutical art. See, e.g. Remington Pharmaceutical Science, 18thEd., Merck Publishing Co. Eastern Pa., (1990). One preferred preparationutilizes a vehicle of physiological saline solution, but it iscontemplated that other pharmaceutically acceptable carriers such asphysiological concentrations of other non-toxic salts, five percentaqueous glucose solution, sterile water or the like may also be used. Itmay also be desirable that a suitable buffer be present in thecomposition. Such solutions can, if desired, be lyophilized and storedin a sterile ampoule ready for reconstitution by the addition of sterilewater for ready injection. The primary solvent can be aqueous oralternatively non-aqueous. The subject prostate tumor antigens,fragments or variants thereof can also be incorporated into a solid orsemi-solid biologically compatible matrix which can be implanted intotissues requiring treatment.

The carrier can also contain other pharmaceutically-acceptableexcipients for modifying or maintaining the pH, osmolarity, viscosity,clarity, color, sterility, stability, rate of dissolution, or odor ofthe formulation. Similarly, the carrier may contain still otherpharmaceutically-acceptable excipients for modifying or maintainingrelease or absorption or penetration across the blood-brain barrier.Such excipients are those substances usually and customarily employed toformulate dosages for parental administration in either unit dosage ormulti-dose form or for direct infusion into the cerebrospinal fluid bycontinuous or periodic infusion.

Dose administration can be repeated depending upon the pharmacokineticparameters of the dosage formulation and the route of administrationused.

It is also contemplated that certain formulations containing the subjectantibody or nucleic acid antagonists are to be administered orally. Suchformulations are preferably encapsulated and formulated with suitablecarriers in solid dosage forms. Some examples of suitable carriers,excipients, and diluents include lactose, dextrose, sucrose, sorbitol,mannitol, starches, gum acacia, calcium phosphate, alginates, calciumsilicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose,gelatin, syrup, methyl cellulose, methyl- and propylhydroxybenzoates,talc, magnesium, stearate, water, mineral oil, and the like. Theformulations can additionally include lubricating agents, wettingagents, emulsifying and suspending agents, preserving agents, sweeteningagents or flavoring agents. The compositions may be formulated so as toprovide rapid, sustained, or delayed release of the active ingredientsafter administration to the patient by employing procedures well knownin the art. The formulations can also contain substances that diminishproteolytic degradation and promote absorption such as, for example,surface active agents.

The specific dose is calculated according to the approximate body weightor body surface area of the patient or the volume of body space to beoccupied. The dose will also be calculated dependent upon the particularroute of administration selected. Further refinement of the calculationsnecessary to determine the appropriate dosage for treatment is routinelymade by those of ordinary skill in the art. Such calculations can bemade without undue experimentation by one skilled in the art in light ofthe activity disclosed herein in assay preparations of target cells.Exact dosages are determined in conjunction with standard dose-responsestudies. It will be understood that the amount of the compositionactually administered will be determined by a practitioner, in the lightof the relevant circumstances including the condition or conditions tobe treated, the choice of composition to be administered, the age,weight, and response of the individual patient, the severity of thepatient's symptoms, and the chosen route of administration.

In one embodiment of this invention, the protein may be therapeuticallyadministered by implanting into patients vectors or cells capable ofproducing a biologically-active form of the protein or a precursor ofprotein, i.e., a molecule that can be readily converted to abiological-active form of the protein by the body. In one approach,cells that secrete the protein may be encapsulated into semipermeablemembranes for implantation into a patient. The cells can be cells thatnormally express the protein or a precursor thereof or the cells can betransformed to express the protein or a precursor thereof It ispreferred that the cell be of human origin and that the protein be ahuman protein when the patient is human. However, it is anticipated thatnon-human primate homologues of the protein discussed infra may also beeffective.

Detection of Subject Prostate Proteins or Nucleic Acids

In a number of circumstances it would be desirable to determine thelevels of protein or corresponding mRNA in a patient. Evidence disclosedinfra suggests the subject prostate proteins may be expressed atdifferent levels during some diseases, e.g., cancers, provides the basisfor the conclusion that the presence of these proteins serves a normalphysiological function related to cell growth and survival. Endogenouslyproduced protein according to the invention may also play a role incertain disease conditions.

The term “detection” as used herein in the context of detecting thepresence of protein in a patient is intended to include the determiningof the amount of protein or the ability to express an amount of proteinin a patient, the estimation of prognosis in terms of probable outcomeof a disease and prospect for recovery, the monitoring of the proteinlevels over a period of time as a measure of status of the condition,and the monitoring of protein levels for determining a preferredtherapeutic regimen for the patient, e.g. one with prostate cancer.

To detect the presence of a prostate protein according to the inventionin a patient, a sample is obtained from the patient. The sample can be atissue biopsy sample or a sample of blood, plasma, serum, CSF, urine orthe like. It has been found that the subject proteins are expressed athigh levels in some cancers. Samples for detecting protein can be takenfrom prostate tissues. When assessing peripheral levels of protein, itis preferred that the sample be a sample of blood, plasma or serum. Whenassessing the levels of protein in the central nervous system apreferred sample is a sample obtained from cerebrospinal fluid or neuraltissue. The sample may be obtained by non-invasive methods, such as fromtissue collection(s) or cultute(s), or using directly available tissuematerial (urine, saliva, stools, hair, etc.).

In some instances, it is desirable to determine whether the gene isintact in the patient or in a tissue or cell line within the patient. Byan intact gene, it is meant that there are no alterations in the genesuch as point mutations, deletions, insertions, chromosomal breakage,chromosomal rearrangements and the like wherein such alteration mightalter production of the corresponding protein or alter its biologicalactivity, stability or the like to lead to disease processes. Thus, inone embodiment of the present invention a method is provided fordetecting and characterizing any alterations in the gene. The methodcomprises providing an oligonucleotide that contains the gene, genomicDNA or a fragment thereof or a derivative thereof. By a derivative of anoligonucleotide, it is meant that the derived oligonucleotide issubstantially the same as the sequence from which it is derived in thatthe derived sequence has sufficient sequence complementarily to thesequence from which it is derived to hybridize specifically to the gene.The derived nucleotide sequence is not necessarily physically derivedfrom the nucleotide sequence, but may be generated in any mannerincluding for example, chemical synthesis or DNA replication or reversetranscription or transcription.

Typically, patient genomic DNA is isolated from a cell sample from thepatient and digested with one or more restriction endonucleases such as,for example, TaqI and AluI. Using the Southern blot protocol, which iswell known in the art, this assay determines whether a patient or aparticular tissue in a patient has an intact prostate gene according tothe invention or a gene abnormality.

Hybridization to a gene would involve denaturing the chromosomal DNA toobtain a single-stranded DNA; contacting the single-stranded DNA with agene probe associated with the gene sequence; and identifying thehybridized DNA-probe to detect chromosomal DNA containing at least aportion of a gene.

The term “probe” as used herein refers to a structure comprised of apolynucleotide that forms a hybrid structure with a target sequence, dueto complementarily of probe sequence with a sequence in the targetregion. Oligomers suitable for use as probes may contain a minimum ofabout 8-12 contiguous nucleotides which are complementary to thetargeted sequence and preferably a minimum of about 20.

A gene according to the present invention can be DNA or RNAoligonucleotides and can be made by any method known in the art such as,for example, excision, transcription or chemical synthesis. Probes maybelabeled with any detectable label known in the art such as, for example,radioactive or fluorescent labels or enzymatic marker. Labeling of theprobe can be accomplished by any method known in the art such as by PCR,random priming, end labeling, nick translation or the like. One skilledin the art will also recognize that other methods not employing alabeled probe can be used to determine the hybridization. Examples ofmethods that can be used for detecting hybridization include Southernblotting, fluorescence in situ hybridization, and single-strandconformation polymorphism with PCR amplification.

Hybridization is typically carried out at 250-45° C., more preferably at32°-40° C. and more preferably at 37°-38° C. The time required forhybridization is from about 0.25 to about 96 hours, more preferably fromabout one to about 72 hours, and most preferably from about 4 to about24 hours.

Gene abnormalities can also be detected by using the PCR method andprimers that flank or lie within the gene. The PCR method is well knownin the art. Briefly, this method is performed using two oligonucleotideprimers which are capable of hybridizing to the nucleic acid sequencesflanking a target sequence that lies within a gene and amplifying thetarget sequence. The terms “oligonucleotide primer” as used hereinrefers to a short strand of DNA or RNA ranging in length from about 8 toabout 30 bases. The upstream and downstream primers are typically fromabout 20 to about 30 base pairs in length and hybridize to the flankingregions for replication of the nucleotide sequence. The polymerizationis catalyzed by a DNA-polymerase in the presence of deoxynucleotidetriphosphates or nucleotide analogs to produce double-stranded DNAmolecules. The double strands are then separated by any denaturingmethod including physical, chemical or enzymatic. Commonly, a method ofphysical denaturation is used involving heating the nucleic acid,typically to temperatures from about 80° C. to 105° C. for times rangingfrom about 1 to about 10 minutes. The process is repeated for thedesired number of cycles.

The primers are selected to be substantially complementary to the strandof DNA being amplified. Therefore, the primers need not reflect theexact sequence of the template, but must be sufficiently complementaryto selectively hybridize with the strand being amplified.

After PCR amplification, the DNA sequence comprising the gene or afragment thereof is then directly sequenced and analyzed by comparisonof the sequence with the sequences disclosed herein to identifyalterations which might change activity or expression levels or thelike.

In another embodiment, a method for detecting a tumor protein accordingto the invention is provided based upon an analysis of tissue expressingthe gene. Certain tissues such as prostate tissues have been found tooverexpress the subject gene. The method comprises hybridizing apolynucleotide to mRNA from a sample of tissue that normally expressesthe gene. The sample is obtained from a patient suspected of having anabnormality in the gene.

To detect the presence of mRNA encoding the protein, a sample isobtained from a patient. The sample can be from blood or from a tissuebiopsy sample. The sample may be treated to extract the nucleic acidscontained therein. The resulting nucleic acid from the sample issubjected to gel electrophoresis or other size separation techniques.

The mRNA of the sample is contacted with a DNA sequence serving as aprobe to form hybrid duplexes. The use of a labeled probes as discussedabove allows detection of the resulting duplex.

When using the cDNA encoding the protein or a derivative of the cDNA asa probe, high stringency conditions can be used in order to preventfalse positives, that is the hybridization and apparent detection of thegene nucleotide sequence when in fact an intact and functioning gene isnot present. When using sequences derived from the gene cDNA, lessstringent conditions could be used, however, this would be a lesspreferred approach because of the likelihood of false positives. Thestringency of hybridization is determined by a number of factors duringhybridization and during the washing procedure, including temperature,ionic strength, length of time and concentration of formamide. Thesefactors are outlined in, for example, Sambrook et al. [Sambrook et al.(1989), supra].

In order to increase the sensitivity of the detection in a sample ofmRNA encoding the protein A or protein B, the technique of reversetranscription/polymerization chain reaction (RT/PCR) can be used toamplify cDNA transcribed from mRNA encoding the prostate tumor antigen.The method of RT/PCR is well known in the art, and can be performed asfollows. Total cellular RNA is isolated by, for example, the standardguanidium isothiocyanate method and the total RNA is reversetranscribed. The reverse transcription method involves synthesis of DNAon a template of RNA using a reverse transcriptase enzyme and a 3′ endprimer. Typically, the primer contains an oligo(dT) sequence. The cDNAthus produced is then amplified using the PCR method and gene A or geneB specific primers. [Belyavsky et al., Nucl. Acid Res. 17:2919-2932(1989); Krug and Berger, Methods in Enzymology, 152:316-325, AcademicPress, NY (1987) which are incorporated by reference].

The polymerase chain reaction method is performed as described aboveusing two oligonucleotide primers that are substantially complementaryto the two flanking regions of the DNA segment to be amplified.Following amplification, the PCR product is then electrophoresed anddetected by ethidium bromide staining or by phosphoimaging.

The present invention further provides for methods to detect thepresence of the protein in a sample obtained from a patient. Any methodknown in the art for detecting proteins can be used. Such methodsinclude, but are not limited to immunodiffusion, immunoelectrophoresis,immunochemical methods, binder-ligand assays, immunohistochemicaltechniques, agglutination and complement assays. [Basic and ClinicalImmunology, 217-262, Sites and Terr, eds., Appleton & Lange, Norwalk,Conn., (1991),which is incorporated by reference]. Preferred arebinder-ligand immunoassay methods including reacting antibodies with anepitope or epitopes of the prostate tumor antigen protein andcompetitively displacing a labeled prostate antigen according to theinvention or derivative thereof.

As used herein, a derivative of the subject prostate tumor antigen isintended to include a polypeptide in which certain amino acids have beendeleted or replaced or changed to modified or unusual amino acidswherein the derivative is biologically equivalent to gene and whereinthe polypeptide derivative cross-reacts with antibodies raised againstthe protein. By cross-reaction it is meant that an antibody reacts withan antigen other than the one that induced its formation.

Numerous competitive and non-competitive protein binding immunoassaysare well known in the art. Antibodies employed in such assays maybeunlabeled, for example as used in agglutination tests, or labeled foruse in a wide variety of assay methods. Labels that can be used includeradionuclides, enzymes, fluorescers, chemiluminescers, enzyme substratesor co-factors, enzyme inhibitors, particles, dyes and the like for usein radioinununoassay (RIA), enzyme immnunoassays, e.g., enzyme-linkedimmunosorbent assay (ELISA), fluorescent immunoassays and the like.

A further aspect of this invention relates to a method for selecting,identifying, screening, characterizing or optimizing biologically activecompounds, comprising a determination of whether a candidate compoundbinds, preferably selectively, a target molecule as disclosed above.Such target molecules include nucleic acid sequences, polypeptides andfragments thereof, typically prostate-specific antigens, even morepreferably extracellular portions thereof. Binding may be assessed invitro or in vivo, typically in vitro, in cell based or accellularsystems. Typically, the target molecule is contacted with the candidatecompound in any appropriate device, and the formation of a complex isdetermined. The target molecule and/or the candidate compound maybeimmobilized on a support. The compounds identified or selected representdrug candidates or leads for treating cancer diseases, particularlyprostate cancer.

While the invention has been described supra, including preferredembodiments, the following examples are provided to further illustratethe invention.

EXAMPLE

Tissue Sources:

Appropriate patient samples were procured for evaluation of researchprotocol. Samples were provided with relevant clinical parameters, andpatient consent. Histological assessment was performed on all samplesand diagnosis by pathology confirmed the presence and/or absence ofmalignancy within each sample. Clinical data generally included patenthistory, physiopathology, and parameters relating to prostate cancerphysiology. Ten normal and ten malignant samples were procured alongwith available clinical information. In addition, ten samples fromorgans other than normal prostate and prostate cancer were procured todetermine the tissue specific expression profile of epitopes. RNAderived from normal tissue samples was obtained from known commercialsources.

Generation of the DATAS Library

Samples were pooled based on their pathological diagnosis (normal vs.tumor). Samples were pooled based on equivalent amounts of total RNA toproduce total pooled RNA samples of 100 ug. DATAS libraries wereconstructed as previously disclosed in U.S. Pat. No. 6,251,590, thedisclosure of which is incorporated by reference in its entirety.Briefly, total RNA was isolated from the normal and tumor pooled samplesand mRNA was subsequently purified from the total RNA for each pooledsample. Synthesis of cDNA was performed using a biotinylated oligo (dT)primer. The biotinylated cDNA was hybridized with the mRNA of theopposite sample to form heteroduplexes between the cDNA and the mRNA.For example, the biotinylated cDNA of the pooled normal prostate samplewas hybridized with prostate tumor mRNA. Similarly, prostate tumorbiotinylated cDNA was hybridized with prostate normal RNA to generatethe second DATAS library. Streptavidin coated beads were used to purifythe complexes by binding the biotin present on the cDNA. Theheteroduplexes were digested with RNAse H to degrade the RNA that wascomplementary to the cDNA. All mRNA sequences that were different fromthe cDNA remained intact. These single stranded RNA fragments or “loops”were subsequently amplified with degenerate primers and cloned intoeither pGEM-Tor pCR II TOPO vector (Company source) to produce the DATASlibrary.

Clone sequencing and Bioinformatics Analysis:

The DATAS library was used to transform E. Coli so that individualclones could be isolated using standard molecular biology techniques.From these libraries, 10,665 individual clones were isolated andsequenced using an automated Applied Biosystems 3100 sequencer. Thenucleotide sequences that were obtained were submitted to thebioinformatics pipeline for analysis. As the DATAS library is preparedwith PCR amplified DNA, many copies of the same sequence are present inthe clones isolated from the libraries. Therefore it is important toreduce the redundancy of the clones to identify the number of unique,nonrepeating sequences that are isolated. From this large set of DATASfragments, 1699 unique, nonredundant sequences were identified and eachDATAS fragment was annotated with a candidate gene. The annotation wasperformed by aligning the DATAS fragment to the human genome sequence bytwo methods; 1) a publicly available alignment and genome viewer tool,Blat (Kent et al., 2002); and 2) a commercially available genomicalignment andviewer tool, Prophecy (Doubletwist). Each DATAS fragmentsequence was annotated with a corresponding gene that overlapped thegenomic sequence containing the DATAS fragment. Genes were annotatedwith either the RefSeq accession number, or a hypothetical geneprediction from different algorithms, for example, Genscan, Twinscan, orFgenesh++. Identified genes were either matched to the sequence of theDATAS fragment (in case of exon to fragment match), or overlapped withthe DATAS fragment (in case of intron to fragment match), and the fulllength sequence of the gene was identified. These sequences were furtheranalyzed to detect all potential membrane spanning proteins. Membraneproteins were predicted through the use of different algorithms publiclyavailable. For example, TMHMM (CBS) was used to identifymembrane-spanning domains present within the amino acid sequence of thecandidate gene. DATAS fragments were located within the sequence in anattempt to determine whether the spliced event affected intracellular orextraceullar domains. Genes associated with the sequence were ranked inorder to maximize the identification of successful therapeutic targets.The highest priority genes had characteristics where the gene was aknown membrane protein, the function of the gene was known, and theDATAS fragment mapped to an intron on the extracellular domain of theprotein, indicating that the DATAS fragment would be presented outsidethe cell, and available for therapeutic intervention by monoclonalantibodies.

Based on the bioinformatic analysis, clones were prioritized in threegroups:

-   -   A) Known transmembrane genes with DATAS fragments located in        introns on the extracellular domain.    -   B) Known and predicted transmembrane genes with DATAS fragments        located in exons in either the extracellular or intracellular        domain.    -   C) DATAS fragments that did not match the genome        Expression Monitoring:

A valid epitope target for prostate cancer requires that the expressionof the epitope be limited to prostate tissue, or preferably to prostatetumors. Assessment of the expression profile for each prioritizedsequence was performed by RT-PCR, a procedure well known in the art. Aprotocol known as touchdown PCR was used, described in the user's manualfor the GeneAmp PCR system 9700, Applied Biosystems. Briefly, PCRprimers were designed to the DATAS fragment and used for end pointRT-PCR analysis. Each RT reaction contained 5 μg of total RNA and wasperformed in a 100 μl volume using Archive RT Kit (Applied Biosystems).The RT reactions were diluted 1:50 with water and 4 μl of the dilutedstock was used in a 50 μl PCR reaction consisting of one cycle at 94° C.for 3 min, 5 cycles at 94° C. for 30 seconds, 60° C. for 30 seconds and72° C. for 45 seconds, with each cycle reducing the annealingtemperature by 0.5 degree. This was followed by 30 cycles at 94° C. for30 seconds, 55° C. for 30 seconds, and 72° C. for 45 seconds. 15 μl wasremoved from each reaction for analysis and the reactions were allowedto proceed for an additional 10 cycles. This produced reactions foranalysis at 30 and 40 cycles, and allowed the detection of differencesin expression where the 40 cycle reactions had saturated. The level ofexpression profile of the DATAS fragment was determined in normal andtumor prostate total RNA, as well as total RNA from normal samples ofbrain, heart, liver, lung, kidney, colon, bone marrow, muscle, spleen,and testis. Expression profiles were prioritized accordingly forspecific expression in prostate tumor and low expression found in normaltissues, including normal prostate.

Verification of RNA Structure:

DATAS identifies sequences that are altered between the experimentalsamples. However, the exact sequence of the junctions or borders thatthe DATAS fragment represents can not be determined directly from theisolated DATAS fragment sequence. The DATAS fragment was used, however,to design experiments that elucidate the sequence of each transcriptpresent in each sample. Primers were designed to amplify a region of thegene larger than the proposed DATAS fragment sequence. These ampliconswere subsequently cloned and sequenced for the identification of theexact junctions of all exons and introns. This required partial cloningof the isoforms from an identified sample to verify the primarystructure (sequence) of the isoforms. All twenty samples (10 normal and10 tumor samples) initially used to generate the DATAS libraries wereused for the verification of the mRNA structure of the prioritizedgenes.

Isolation of Full-Length Clones of Isoforms:

Isolation of the full-length clones containing both isoforms wasaccomplished utilizing the information and DNA fragments generatedduring the structure validation process. Several methods are applicableto isolation of the full length clone. Where full sequence informationregarding the coding sequence is available, gene specific primers weredesigned from the sequence and used to amplify the coding sequencedirectly from the total RNA of the tissue samples. An RT-PCR reactionwas set up using these gene specific primers. The RT reaction wasperformed as described infra, using oligo dT to prime for cDNA. Secondstrand was produced by standard methods to produce double stranded cDNA.PCR amplification of the gene was accomplished using gene specificprimers. PCR consisted of 30 cycles at 94° C. for 30 seconds, 55° C. for30 seconds, and 72° C. for 45 seconds. The reaction products wereanalyzed on 1% agarose gels and the amplicons were ligated into preparedvectors with A overhangs for amplicon cloning. 1 μl of the ligationmixture was used to transform E. Coli for cloning and isolation of theamplicon. Once purified, the plasmid containing the amplicon wassequenced on an ABI 3100 automated sequencer.

Where limited sequence information was available, the oligo pullingmethod was utilized. Briefly, a gene-specific oligonucleotide wasdesigned based on the DATAS fragment. The oligonucleotide was labeledwith biotin and used to hybridize with a single stranded plasmid DNAlibrary prepared from either normal prostate tissue or prostate tumortissue following the procedures of Sambrook et al (1989). The hybridizedcDNA was separated by streptavidin conjugated beads and eluted byheating. The eluted cDNA was converted to double strand plasmid DNA andused to transform E. Coli cells and the longest cDNA clone was subjectedto DNA sequencing.

RESULTS

Using methods described above, 1699 DNA fragments have been identifiedthat putatively correspond to exons (novel splice variants) expressedexclusively or at an increased level in prostate tumor tissue whencompared to matched normal prostate tissue.

These sequences were used to search public databases containing humangenomic sequences to identify related genes. This search identified 122fragments that correspond to exons of either known or potential cellsurface proteins.

Additionally, thirty seven distinct alternatively spliced isoforms wereidentified from the initial sequence tags that appear to contain novelsequence information of cell surface proteins.

These DNA sequences are disclosed in the Sequence Listing as well as inTable 1, and correspond to the nucleic acid sequences having SEQ ID NOS:1-173, 175, 177, 179, and 181. Oligonucleotide primers were designed toeach DATAS fragment to determine the specific expression of the mRNA ina panel of normal human tissue. An example is shown in FIG. 1, where theclone corresponding to Sequence ID: No.92 displays specific expressionin prostate with very low levels detected in kidney (lane 4) andpancreas (lane 9). All clones that were found to be either specificallyexpressed in prostate or highly expressed in prostate compared to othertissues were analyzed for expression in tumor samples.

FIG. 2 illustrates the expression profile of one DATAS clone in normaland tumor prostate tissue. Expression of this clone is upregulated intwo of the three tumor pooled samples and is highly expressed in threeof the four individual tumor samples. The high expression of this spliceevent in tumor samples as compared to normal prostate, and the lowexpression in other normal human tissues is an example of one candidatethat has utility for development as a novel epitope for prostate cancer.

The splice events for DATAS clones that displayed a specific expressionprofile for prostate and a high differential expression profile forprostate tumors were isolated and the sequences for each event wasdetermined. An example is shown in FIG. 3, where the sequence of theisolated event was mapped to the genome in Blat, and genomic viewerdeveloped by the bioinformatics department at UCSC (Kent et al., 2002).Five distinct clones were isolated that mapped to the gene locus forSTEAP2. One expressed sequence tag (EST), AK092666, contained manysimilar domains as the splice events that were isolated using DATAS. Thesequences and predicted protein translations for all five clones aredescribed in SEQ NOS. 173-182 and are graphically illustrated in FIG. 3.The length of the open reading frame and the predicted protein size foreach isoform is described in Table 2. The EST, AK092666 contains a largedeletion in exon 5, the terminal 3′ exon of STEAP2, with two novel exonsin the 5′ region of the transcript. The nomenclature for the DATASderived events was based on AK092666 because of higher similarity whencompared to the RefSeq sequence for STEAP2. The first isoformidentified, AK092666_(—)01 (SEQ ID NO 173), contains a novel C-terminalexon when compared to AK092666, and therefore generates a noveljunction, and a novel sequence for translation and generates a uniqueamino acid sequence (SEQ ID NO 183). The same novel sequence wasgenerated by isoform AK092666_(—)03 (SEQ ID NO 177), which contains thesame novel exon with an additional splicing event of an in frametruncation of exon 4, and by isoform AK092666_(—)05 (SEQ ID NO 181),which contains a single codon deletion from AK092666_(—)01.AK092666_(—)02 (SEQ ID NO 175) skipped exon 6 of AK092666 and generatedthe novel amino acid sequence in SEQ ID NO 184. AK092666_(—)04 (SEQ IDNO 179) contains a short out of frame truncation of exon 4, whichresults in the creation of 8 novel amino acids before encountering apremature stop codon (SEQ ID NO 185). TABLE 2 Length of the open readingframe and the predicted protein size for each novel isoform. Clone NameORF length (bp) Protein size (KD) STEAP2 1473 56 AK092666 1365 51.7AK092666_01 1389 52.7 AK092666_02 1260 47.8 AK092666_03 900 34.1AK092666_04 705 26.7 AK092666_05 1386 52.5

The novel amino acids found in SEQ ID NOS 183 and 184 represent novelepitopes that are specifically expressed in prostate cancer in amembrane protein. These epitopes are targets for monoclonal antibodyimmunotherapy for the treatment of prostate cancer. To illustrate thedifferent isoforms present, an antibody was generated from the invariantsequence present in the 5′ region (or the amino terminal portion of theprotein) that recognizes all the different isoforms.

An antibody was generated against an amino acid sequence that was commonto all five isoforms, as well as present in STEAP2 and AK092666.Prostate cancer cell lines were analyzed by western blot to determinewhat different isoforms would be expressed at the protein level. FIG. 4illustrates two bands that were specifically detected by the antibody.Band A potentially represents the glycosylated, wild type STEAP2 andband B indicates isoforms AK092666, AK092666_(—)01, or AK092666_(—)05,which is unresolvable in the gel analysis. In addition, multiple bandsof the proper size were detected suggesting that isoforms of the STEAP2locus are expressed and represent targets for the immunotherapy inprostate cancer. TABLE 1 Sequence information of the DATAS fragments andthe alternatively spliced isoforms. Sequence ID: No. 1 Accession #:NM_005656 Genomic sequence: chr21: 39407238-39450894 Sequencedefinition: transmembrane protease serine 2 Sequence ID: No. 2 Accession#: NM_001423 Genomic sequence: chr12: 13265134-13265266 Sequencedefinition: Homo sapiens epithelial membrane protein 1 EMP1 Sequence ID:No. 3 Accession #: NM_000484 Genomic sequence: chr21: 23832850-24123073Sequence definition: beta amyloid A4 Sequence ID: No. 4 Accession #:NM_002841 Genomic sequence: chr3: 62548596-63240788_1 Sequencedefinition: protein tyrosine phosphatase G-type Sequence ID: No. 5Accession #: NM_022124 Genomic sequence: chr10: 74968313-75112962Sequence definition: cadherin related 23 isoform 1 precursor SequenceID: No. 6 Accession #: NM_033056 Genomic sequence: chr10:55940286-56920530_02 Sequence definition: protocadherin 15 precursorSequence ID: No. 7 Accession #: NM_002847 Genomic sequence: chr7:158586667-159621018_01 Sequence definition: protein tyrosine phosphatasereceptor type N Sequence ID: No. 8 Accession #: NM_002222 Genomicsequence: chr3: 5000696-5354641_1 Sequence definition: ITPR inositol145-triphosphate receptor type 1 Sequence ID: No. 9 Accession #:AC078864.20 Genomic sequence: chr12: 52201280-52201714 Sequencedefinition: Genscan prediction Sequence ID: No. 10 Accession #:NM_014554; NM_001844; NT_009785.3 Genomic sequence: chr12:45785273-45856561 Sequence definition: chr12_498 potential fusion ofSENP1 and Collagen 2A; also overlaps GS perdiction Sequence ID: No. 11Accession #: AB064665 Genomic sequence: chrM: 9411-9524 Sequencedefinition: Homo sapiens mRNA for OK/SW-CL.16 Sequence ID: No. 12Accession #: NM_024029 Genomic sequence: chr19: 10880041-10883719Sequence definition: hypothetical protein MGC3262 Sequence ID: No. 13Accession #: NT_008748.79 Genomic sequence: chr10: 80881918-80882092Sequence definition: Genscan prediction Sequence ID: No. 14 Accession #:AB002360 Genomic sequence: chr13: 112761227-112761344 Sequencedefinition: KIAA0362 Sequence ID: No. 15 Accession #: AK057572 Genomicsequence: chr16: 14547315-14547422 Sequence definition: FLJ33010Sequence ID: No. 16 Accession #: NT_034410.56/NM_033102.1 Genomicsequence: chr1: 203503646-203554883/chr1: 192169879-192474008 Sequencedefinition: Genscan - Elk4/LOC85414 - Homo sapiens prostein proteinLOC85414 Sequence ID: No. 17 Accession #: NT_019696.29 Genomic sequence:chrx: 64173951-64275396 Sequence definition: Genscan prediction SequenceID: No. 18 Accession #: NT_007834.17 Genomic sequence: chr7:71656530-71727938 Sequence definition: Genscan prediction Sequence ID:No. 19 Accession #: NT_005403.1000 Genomic sequence: chr2:208067141-208067324 Sequence definition: Genscan prediction Sequence ID:No. 20 Accession #: NT_009654.19 Genomic sequence: chr12:116716120-116840364 Sequence definition: Genscan prediction Sequence ID:No. 21 Accession #: AC126564.7 Genomic sequence: chr12:131440407-131440735 Sequence definition: genomic match Sequence ID: No.22 Accession #: NT_006171.64 Genomic sequence: chr4: 172269202-172299375Sequence definition: Genscan prediction Sequence ID: No. 23 Accession #:NM_025149.1 Genomic sequence: chr17: 61361324-61409903 Sequencedefinition: FLJ20920 Sequence ID: No. 24 Accession #: NT_026437.145Genomic sequence: chr14: 72272372-72462407 Sequence definition: Genscanprediction Sequence ID: No. 25 Accession #: NT_030059.13 Genomicsequence: chr10: 103933731-103955924 Sequence definition: Genscanprediction Sequence ID: No. 26 Accession #: AK058112 Genomic sequence:chr19: 1815692-1822319 Sequence definition: FLJ25383 Sequence ID: No. 27Accession #: NM_002205.1 Genomic sequence: chr12: 55541534-55565494Sequence definition: Homo sapiens integrin alpha 5 fibronectin receptoralpha polypeptide Sequence ID: No. 28 Accession #: NM_004716.1 Genomicsequence: chr11: 117114115-117114448 Sequence definition: Homo sapiensproprotein convertase subtilisin/kexin type 7 PCSK7 mRNA Sequence ID:No. 29 Accession #: NM_030774 Genomic sequence: chr11: 5003431-5021099Sequence definition: prostate specific G-protein coupled receptor [Homosapiens] Sequence ID: No. 30 Accession #: AB007932 Genomic sequence:chr1: 204846394-204846755 Sequence definition: Homo sapiens plexin A2PLXNA2 mRNA Sequence ID: No. 31 Accession #: AB023177 Genomic sequence:chr7: 11157196-11157402 Sequence definition: Homo sapiens mRNA forKIAA0960 protein Sequence ID: No. 32 Accession #: NT_004858.23 Genomicsequence: chr1: 147688399-147725025 Sequence definition: Genscanprediction Sequence ID: No. 33 Accession #: NT_004873.61 Genomicsequence: chr1: 14678698-14732191 Sequence definition: Genscanprediction Sequence ID: No. 34 Accession #: NT_029860.99 Genomicsequence: chr1: 110751286-110854188 Sequence definition: Genscanprediction Sequence ID: No. 35 Accession #: NM_032385.1 Genomicsequence: chr5: 170086201-170251515 Sequence definition: Homo sapienschromosome 5 open reading frame 4 C5orf4 Sequence ID: No. 36 Accession#: NM_014752.1 Genomic sequence: chr11: 73182947-73211393 Sequencedefinition: KIAA0102 Sequence ID: No. 37 Accession #: NP_000295 Genomicsequence: chr17: 15500091-15500332 Sequence definition: Homo sapiensperipheral myelin protein 22 Sequence ID: No. 38 Accession #: NM_020433Genomic sequence: chr20: 42528457-42528759 Sequence definition: Homosapiens junctophilin 2 Sequence ID: No. 39 Accession #: NT_999999.2Genomic sequence: chrM: 9411-9524 Sequence definition: Genscan GenePredictions Sequence ID: No. 40 Accession #: NT_004754.1 Genomicsequence: chr1: 117988850-117989247 Sequence definition: Genscan GenePredictions Sequence ID: No. 41 Accession #: NT_011568.108 Genomicsequence: chrX: 47583156-47583796 Sequence definition: Acembly GenePredictions/Genscan Gene Predictions Sequence ID: No. 42 Accession #:NP_061116 Genomic sequence: chr7: 140900079-140900876 Sequencedefinition: transient receptor potential cation channel Sequence ID: No.43 Accession #: NT_011295.163 Genomic sequence: chr19: 19799239-19804450Sequence definition: Genscan prediction Sequence ID: No. 44 Accession #:NP_056051 Genomic sequence: chr4: 62284401-62284770 Sequence definition:lectomedin-3 Sequence ID: No. 45 Accession #: NT_033927.57 Genomicsequence: chr11: 75518014-75562375 Sequence definition: Genscanprediction Sequence ID: No. 46 Accession #: NM_030774 Genomic sequence:chr11: 5003431-5021099 Sequence definition: prostate specific G-proteincoupled receptor Homo sapiens Sequence ID: No. 47 Accession #: NM_022119Genomic sequence: chr16: 2939532-2939842 Sequence definition: proteaseserine 22 Sequence ID: No. 48 Accession #: NP_000155 Genomic sequence:chr19: 46824678-46824801 Sequence definition: Homo sapiens gastricinhibitory polypeptide receptor Sequence ID: No. 49 Accession #:NM_001627 Genomic sequence: chr3: 104784804-104787209 Sequencedefinition: activated leukocyte cell adhesion molecule Sequence ID: No.50 Accession #: NP_056343 Genomic sequence: chr17: 5263335-5263632Sequence definition: Homo sapiens DKFZP566H073 protein Sequence ID: No.51 Accession #: NT_033275.9 Genomic sequence: chr15: 19767754-19767842Sequence definition: Acembly Gene Predictions/Genscan Gene PredictionsSequence ID: No. 52 Accession #: NT_004511.105 Genomic sequence: chr1:37657082-37657508 Sequence definition: Genscan Gene Predictions SequenceID: No. 53 Accession #: NT_007819.76 Genomic sequence: chr7:2293638-2293859 Sequence definition: Acembly Gene Predictions/GenscanGene Predictions Sequence ID: No. 54 Accession #: NT_008046.179 Genomicsequence: chr8: 101509107-101509191 Sequence definition: Acembly GenePredictions/Genscan Gene Predictions Sequence ID: No. 55 Accession #:NP_001668 Genomic sequence: chr1: 166712801-166712951 Sequencedefinition: ATPase Na+/K+ transporting beta 1 polypeptide Sequence ID:No. 56 Accession #: NP_061332 Genomic sequence: chr7:105724807-105753208 Sequence definition: B-cell receptor-associatedprotein BAP29 Sequence ID: No. 57 Accession #: NT_008251.42 Genomicsequence: chr8: 36531104-36531405 Sequence definition: Acembly GenePredictions/Genscan Gene Predictions Sequence ID: No. 58 Accession #:NT_008984.116 Genomic sequence: chr11: 97792879-97792961 Sequencedefinition: Genscan Gene Predictions Sequence ID: No. 59 Accession #:NT_011176.84 Genomic sequence: chr19: 11151260-11154382 Sequencedefinition: Acembly Gene Predictions/Genscan Gene Predictions SequenceID: No. 60 Accession #: ENST00000255124 Genomic sequence: chr20:46047371-46047445 Sequence definition: Acembly Gene Predictions/GenscanGene Predictions Sequence ID: No. 61 Accession #: ENST00000262657Genomic sequence: chr20: 29935469-29937596 Sequence definition: AcemblyGene Predictions/Genscan Gene Predictions Sequence ID: No. 62 Accession#: NT_033903.44 Genomic sequence: chr11: 58671001-58671164 Sequencedefinition: Genscan Gene Predictions Sequence ID: No. 63 Accession #:NP_000360 Genomic sequence: chr14: 78989775-78989913 Sequencedefinition: Homo sapiens thyroid stimulating hormone receptor SequenceID: No. 64 Accession #: NP_005219 Genomic sequence: chr7:54724858-54725037 Sequence definition: Homo sapiens epidermal growthfactor receptor erythroblastic leukemia viral v-erb-b oncogene homologavian Sequence ID: No. 65 Accession #: NP_149093 Genomic sequence: chr1:203548697-203549088 Sequence definition: Homo sapiens prostein proteinSequence ID: No. 66 Accession #: NM_030774 Genomic sequence: chr11:5003431-5021099 Sequence definition: prostate specific G-protein coupledreceptor Homo sapiens Sequence ID: No. 67 Accession #: NM_030774 Genomicsequence: chr11: 5004995-5010301 Sequence definition: prostate specificG-protein coupled receptor Homo sapiens Sequence ID: No. 68 Accession #:NM_030774 Genomic sequence: chr11: 5004983-5010305 Sequence definition:prostate specific G-protein coupled receptor Homo sapiens Sequence ID:No. 69 Accession #: NM_030774 Genomic sequence: chr11: 5004983-5010305Sequence definition: prostate specific G-protein coupled receptor Homosapiens Sequence ID: No. 70 Accession #: NM_030774 Genomic sequence:chr11: 4667240-4678100 Sequence definition: prostate specific G-proteincoupled receptor Homo sapiens Sequence ID: No. 71 Accession #: NM_030774Genomic sequence: chr11: 4677792-4677987 Sequence definition: prostatespecific G-protein coupled receptor Homo sapiens Sequence ID: No. 72Accession #: NM_030774 Genomic sequence: chr11: 5003430-5007773 Sequencedefinition: prostate specific G-protein coupled receptor Homo sapiensSequence ID: No. 73 Accession #: AK075546 Genomic sequence: chr11:36643617-36930167 Sequence definition: predicted protein Sequence ID:No. 74 Accession #: AK075546 Genomic sequence: chr11: 36643626-36931023Sequence definition: predicted protein Sequence ID: No. 75 Accession #:AK075546 Genomic sequence: chr11: 36643617-36929351 Sequence definition:predicted protein Sequence ID: No. 76 Accession #: AK075546 Genomicsequence: chr11: 36643617-36929351 Sequence definition: predictedprotein Sequence ID: No. 77 Accession #: AK075546 Genomic sequence:chr11: 36643617-36929351 Sequence definition: predicted protein SequenceID: No. 78 Accession #: NT_033927.57 Genomic sequence: chr11:75518014-75562375 Sequence definition: Genscan prediction Sequence ID:No. 79 Accession #: NM_000300 Genomic sequence: chr1: 19337078-19342056Sequence definition: phospholipase A2 group IIA platelets synovialSequence ID: No. 80 Accession #: NM_000300 Genomic sequence: chr1:19337078-19342056 Sequence definition: phospholipase A2 group IIAplatelets synovial Sequence ID: No. 81 Accession #: NM_000300 Genomicsequence: chr1: 19337078-19342056 Sequence definition: phospholipase A2group IIA platelets synovial Sequence ID: No. 82 Accession #: NM_000300Genomic sequence: chr1: 19337078-19342056 Sequence definition:phospholipase A2 group IIA platelets synovial Sequence ID: No. 83Accession #: NM_000300 Genomic sequence: chr1: 19337078-19342056Sequence definition: phospholipase A2 group IIA platelets synovialSequence ID: No. 84 Accession #: NM_032323 Genomic sequence: chr1:152017962-152027457 Sequence definition: hypothetical protein MGC13102 -refseq Sequence ID: No. 85 Accession #: NM_032323 Genomic sequence:chr1: 152017962-152027457 Sequence definition: hypothetical proteinMGC13102 Sequence ID: No. 86 Accession #: NM_032323 Genomic sequence:chr1: 152017962-152027457 Sequence definition: hypothetical proteinMGC13102 Sequence ID: No. 87 Accession #: NM_032323 Genomic sequence:chr1: 152017962-152027457 Sequence definition: hypothetical proteinMGC13102 Sequence ID: No. 88 Accession #: NM_032323 Genomic sequence:chr1: 152017962-152027457 Sequence definition: hypothetical proteinMGC13102 Sequence ID: No. 89 Accession #: NM_032323 Genomic sequence:chr1: 152017962-152027457 Sequence definition: hypothetical proteinMGC13102 Sequence ID: No. 90 Accession #: NM_032323 Genomic sequence:chr1: 152017962-152027457 Sequence definition: hypothetical proteinMGC13102 Sequence ID: No. 91 Accession #: AK092666 Genomic sequence:chr7: 88376306-88402240 Sequence definition: STEAP2/AK092666 SequenceID: No. 92 Accession #: AK092666 Genomic sequence: chr7:88376306-88402240 Sequence definition: STEAP2/AK092666 Sequence ID: No.93 Accession #: AK092666 Genomic sequence: chr7: 88376306-88402240Sequence definition: STEAP2/AK092666 Sequence ID: No. 94 Accession #:NM_005656 Genomic sequence: chr21: 39493446-39537043 Sequencedefinition: TMPRSS2 Sequence ID: No. 95 Accession #: NM_005656 Genomicsequence: chr21: 39493446-39537043 Sequence definition: TMPRSS2 SequenceID: No. 96 Accession #: NM_005656 Genomic sequence: chr21:39493446-39537043 Sequence definition: TMPRSS2 Sequence ID: No. 97Accession #: NM_005656 Genomic sequence: chr21: 39493446-39537043Sequence definition: TMPRSS2 Sequence ID: No. 98 Accession #: NM_005656Genomic sequence: chr21: 39493446-39537043 Sequence definition: TMPRSS2Sequence ID: No. 99 Accession #: NM_004476 Genomic sequence: chr11:50361918-50423952 Sequence definition: PSMA/FOLH1 Sequence ID: No. 100Accession #: NM_004476 Genomic sequence: chr11: 50361918-50423952Sequence definition: PSMA/FOLH1 Sequence ID: No. 101 Accession #:NM_004476 Genomic sequence: chr11: 50361918-50423952 Sequencedefinition: PSMA/FOLH1 Sequence ID: No. 102 Accession #: no match toindex Genomic sequence: No match BLAT Sequence definition: No match BLATSequence ID: No. 103 Accession #: AC105101.8 Genomic sequence: chr18:45441503-45442177 Sequence definition: genomic match Sequence ID: No.104 Accession #: BC043509 Genomic sequence: chr2: 7566735-7567210Sequence definition: genomic match Sequence ID: No. 105 Accession #: nomatch to index Genomic sequence: No match BLAT Sequence definition: Nomatch BLAT Sequence ID: No. 106 Accession #: NT_007914.345 Genomicsequence: chr7: 150965224-150965948 Sequence definition: Genscanprediction Sequence ID: No. 107 Accession #: NM_002474 Genomic sequence:chr16: 15123743-15124024 Sequence definition: smooth muscle myosin heavychain 11 isoform Sequence ID: No. 108 Accession #: no match to indexGenomic sequence: No match BLAT Sequence definition: No match BLATSequence ID: No. 109 Accession #: AL450472.14 Genomic sequence: chrX:132596913-132597349 Sequence definition: genomic match Sequence ID: No.110 Accession #: no match to index Genomic sequence: No match BLATSequence definition: No match BLAT Sequence ID: No. 111 Accession #:NM_024490 Genomic sequence: chr15: 18676827-18681314 Sequencedefinition: ATPase Class V type 10A Sequence ID: No. 112 Accession #:NT_007741.24 Genomic sequence: chr7: 154483727-154484200 Sequencedefinition: Genscan prediction Sequence ID: No. 113 Accession #:NT_010168.1 Genomic sequence: chr14: 100136759-100137109 Sequencedefinition: Genscan prediction Sequence ID: No. 114 Accession #:AK074158 Genomic sequence: chr7: 2347770-2347996 Sequence definition:Homo sapiens mRNA for FLJ00231 protein Sequence ID: No. 115 Accession #:no match to index Genomic sequence: No match BLAT Sequence definition:No match BLAT Sequence ID: No. 116 Accession #: AL549429 Genomicsequence: chr11: 9027915-9028089 Sequence definition: genomic matchSequence ID: No. 117 Accession #: NM_015541 Genomic sequence: chr3:65899978-65900329 Sequence definition: leucine-rich repeats andimmunoglobulin-like Sequence ID: No. 118 Accession #: NM_024897 Genomicsequence: chr1: 151978744-151978881 Sequence definition: hypotheticalprotein FLJ22672 Sequence ID: No. 119 Accession #: NM_006598 Genomicsequence: chr5: 1165896-1168793 Sequence definition: solute carrierfamily 12 potassium/chloride Sequence ID: No. 120 Accession #: NM_021569Genomic sequence: chr9: 131740238-131740388 Sequence definition: NMDAreceptor 1 isoform NR1-2 precursor Sequence ID: No. 121 Accession #:AL445467.6 Genomic sequence: chrX: 15985515-15985779 Sequencedefinition: genomic match Sequence ID: No. 122 Accession #: BM976799Genomic sequence: chr1: 54049149-54049432 Sequence definition:genomic/EST match Sequence ID: No. 123 Accession #: no match to indexGenomic sequence: No match BLAT Sequence definition: No match BLATSequence ID: No. 124 Accession #: NT_007933.414 Genomic sequence: chr7:98285605-98286140 Sequence definition: Genscan prediction Sequence ID:No. 125 Accession #: NM_020428 Genomic sequence: chr19:10964586-10965036 Sequence definition: Homo sapiens CTL2 gene CTL2 mRNASequence ID: No. 126 Accession #: no match to index Genomic sequence: Nomatch BLAT Sequence definition: No match BLAT Sequence ID: No. 127Accession #: NM_006292 Genomic sequence: chr11: 19444265-19444422Sequence definition: Homo sapiens tumor susceptibility gene 101 TSG101mRNA Sequence ID: No. 128 Accession #: NM_052932 Genomic sequence:chr11: 102306433-102306907 Sequence definition: Homo sapiens pro-oncosisreceptor inducing membrane injury gene PORIMIN mRNA Sequence ID: No. 129Accession #: NM_000014 Genomic sequence: chr12: 9416444-9416720 Sequencedefinition: Homo sapiens alpha-2-macroglobulin A2M mRNA Sequence ID: No.130 Accession #: NM_002337 Genomic sequence: chr4: 3426547-3433294Sequence definition: low density lipoprotein-related Sequence ID: No.131 Accession #: AL834445 Genomic sequence: chr20: 23304135-23304477Sequence definition: Homo sapiens mRNA; cDNA DKFZp761J109 Sequence ID:No. 132 Accession #: NM_004986 Genomic sequence: chr14:49879277-49880762 Sequence definition: kinectin 1 Sequence ID: No. 133Accession #: NM_024295 Genomic sequence: chr8: 124092754-124095061Sequence definition: hypothetical protein MGC3067 Sequence ID: No. 134Accession #: AC018457.14 Genomic sequence: chr3: 165236534-165236724Sequence definition: genomic match Sequence ID: No. 135 Accession #:NM_004753 Genomic sequence: chr1: 12208898-12258427 Sequence definition:Homo sapiens short-chain dehydrogenase/reductase 1 SDR1 mRNA SequenceID: No. 136 Accession #: NM_004753 Genomic sequence: chr1:12221576-12258383 Sequence definition: Homo sapiens short-chaindehydrogenase/reductase 1 SDR1 mRNA Sequence ID: No. 137 Accession #:NM_004753 Genomic sequence: chr1: 12221576-12258383 Sequence definition:Homo sapiens short-chain dehydrogenase/reductase 1 SDR1 mRNA SequenceID: No. 138 Accession #: NM_004753 Genomic sequence: chr1:12221576-12258383 Sequence definition: Homo sapiens short-chaindehydrogenase/reductase 1 SDR1 mRNA Sequence ID: No. 139 Accession #:NM_004753 Genomic sequence: chr1: 12221576-12258383 Sequence definition:Homo sapiens short-chain dehydrogenase/reductase 1 SDR1 mRNA SequenceID: No. 140 Accession #: D87438 Genomic sequence: chr16:14996279-15058862 Sequence definition: Human mRNA for KIAA0251 genepartial cds Sequence ID: No. 141 Accession #: D87438 Genomic sequence:chr16: 15018972-15027737 Sequence definition: Human mRNA for KIAA0251gene partial cds Sequence ID: No. 142 Accession #: AB007932 Genomicsequence: chr1: 204843635-205060532 Sequence definition: Homo sapiensplexin A2 long form PLXNA2 mRNA Sequence ID: No. 143 Accession #:AB007932 Genomic sequence: chr1: 204843635-205060532 Sequencedefinition: Homo sapiens plexin A2 long form PLXNA2 mRNA Sequence ID:No. 144 Accession #: AB007932 Genomic sequence: chr1:204843635-205060532 Sequence definition: Homo sapiens plexin A2 longform PLXNA2 mRNA Sequence ID: No. 145 Accession #: AB037745 Genomicsequence: chr1: 108833848-108851509 Sequence definition: Homo sapiensmRNA for KIAA1324 protein partial cds Sequence ID: No. 146 Accession #:AB037745 Genomic sequence: chr1: 108851126-108851424 Sequencedefinition: Homo sapiens mRNA for KIAA1324 protein partial cds SequenceID: No. 147 Accession #: AB037745 Genomic sequence: chr1:108851126-108851424 Sequence definition: Homo sapiens mRNA for KIAA1324protein partial cds Sequence ID: No. 148 Accession #: AB037745 Genomicsequence: chr1: 108851126-108851424 Sequence definition: Homo sapiensmRNA for KIAA1324 protein partial cds Sequence ID: No. 149 Accession #:AB037745 Genomic sequence: chr1: 108851126-108851424 Sequencedefinition: Homo sapiens mRNA for KIAA1324 protein partial cds SequenceID: No. 150 Accession #: NM_002253 Genomic sequence: chr4:55795152-55795458 Sequence definition: Homo sapiens kinase insert domainreceptor a type III receptor tyrosine kinase KDR mRNA Sequence ID: No.151 Accession #: NM_004879 Genomic sequence: chr11: 125479160-125481382Sequence definition: Homo sapiens etoposide induced 2.4 mRNA EI24 mRNASequence ID: No. 152 Accession #: BC041788 Genomic sequence: chr8:144841449-144841809 Sequence definition: Homo sapiens Similar to RIKENcDNA 1110025J15 gene clone MGC: 32881 IMAGE: 4738372 mRNA complete cdsSequence ID: No. 153 Accession #: AB033073 Genomic sequence: chr20:46925235-46925516 Sequence definition: Homo sapiens mRNA for KIAA1247protein partial cds Sequence ID: No. 154 Accession #: NT_011520.136Genomic sequence: chr22: 21548074-21562329 Sequence definition: Genscanprediction Sequence ID: No. 155 Accession #: NM_005581 Genomic sequence:chr19: 49998069-49998792 Sequence definition: Homo sapiens Lutheranblood group Auberger b antigen included LU mRNA Sequence ID: No. 156Accession #: NM_004355 Genomic sequence: chr5: 149769000-149775442Sequence definition: Homo sapiens CD74 antigen invariant polypeptide ofmajor histocompatibility complex class II antigen-associated CD74 mRNASequence ID: No. 157 Accession #: NM_000484 Genomic sequence: chr21:26174980-26175131 Sequence definition: Homo sapiens amyloid beta A4precursor protein protease nexin-II Alzheimer disease APP mRNA SequenceID: No. 158 Accession #: NM_005745 Genomic sequence: chrX:150566783-150575554 Sequence definition: Homo sapiens accessory proteinBAP31 BCAP31 mRNA Sequence ID: No. 159 Accession #: NM_005570 Genomicsequence: chr18: 56780509-56781078 Sequence definition: Homo sapienslectin mannose-binding 1 LMAN1 mRNA Sequence ID: No. 160 Accession #:NT_029218.14 Genomic sequence: chr1: 19080562-19080917 Sequencedefinition: Genscan prediction Sequence ID: No. 161 Accession #:NT_011387.8 Genomic sequence: chr20: 410654-410816 Sequence definition:Genscan prediction Sequence ID: No. 162 Accession #: NM_002587 Genomicsequence: chr5: 141227996-141231527 Sequence definition: Homo sapiensprotocadherin 1 PDCH1 Sequence ID: No. 163 Accession #: NT_035036.5Genomic sequence: chr10: 51263955-51274232 Sequence definition: Genscanprediction Sequence ID: No. 164 Accession #: NM_007176 Genomic sequence:chr14: 74107662-74107815 Sequence definition: Homo sapien Chr 14 openreading frame Sequence ID: No. 165 Accession #: AP000531.1 Genomicsequence: chr22: 14703272-14703359 Sequence definition: poor genomicmatch to repeat Sequence ID: No. 166 Accession #: NM_020182 Genomicsequence: chr20: 56850452-56936716 Sequence definition: Homo sapienstransmembrane prostate androgen induced RNA TMEPAI mRNA Sequence ID: No.167 Accession #: NM_020182 Genomic sequence: chr20: 56850452-56936716Sequence definition: Homo sapiens transmembrane prostate androgeninduced RNA TMEPAI mRNA Sequence ID: No. 168 Accession #: NM_020182Genomic sequence: chr20: 56850452-56936716 Sequence definition: Homosapiens transmembrane prostate androgen induced RNA TMEPAI mRNA SequenceID: No. 169 Accession #: NM_020182 Genomic sequence: chr20:56850452-56936716 Sequence definition: Homo sapiens transmembraneprostate androgen induced RNA TMEPAI mRNA Sequence ID: No. 170 Accession#: NM_020182 Genomic sequence: chr20: 56850452-56936716 Sequencedefinition: Homo sapiens transmembrane prostate androgen induced RNATMEPAI mRNA Sequence ID: No. 171 Accession #: NM_020182 Genomicsequence: chr20: 56850452-56936716 Sequence definition: Homo sapienstransmembrane prostate androgen induced RNA TMEPAI mRNA Sequence ID: No.172 Accession #: NM_020182 Genomic sequence: chr20: 56850452-56936716Sequence definition: Homo sapiens transmembrane prostate androgeninduced RNA TMEPAI mRNA Sequence ID: No. 173 Accession #: AK092666_01Sequence definition: Novel spliced isoform of STEAP2 Sequence ID: No.174 Accession #: AK092666_01 Sequence definition: Protein translation ofnovel spliced isoform of STEAP2 Sequence ID: No. 175 Accession #:AK092666_02 Sequence definition: Novel spliced isoform of STEAP2Sequence ID: No. 176 Accession #: AK092666_02 Sequence definition:Protein translation of novel spliced isoform of STEAP2 Sequence ID: No.177 Accession #: AK092666_03 Sequence definition: Novel spliced isoformof STEAP2 Sequence ID: No. 178 Accession #: AK092666_03 Sequencedefinition: Protein translation of novel spliced isoform of STEAP2Sequence ID: No. 179 Accession #: AK092666_04 Sequence definition: Novelspliced isoform of STEAP2 Sequence ID: No. 180 Accession #: AK092666_04Sequence definition: Protein translation of novel spliced isoform ofSTEAP2 Sequence ID: No. 181 Accession #: AK092666_05 Sequencedefinition: Novel spliced isoform of STEAP2 Sequence ID: No. 182Accession #: AK092666_05 Sequence definition: Novel spliced isoform ofSTEAP2 Sequence ID: No. 183 Accession #: AK092666_01aa Sequencedefinition: Novel amino acids generated by spliced isoforms AK092666_01,AK092666_03, AK092666_05 Sequence ID: No. 184 Accession #: AK092666_02aaSequence definition: Novel amino acids generated by spliced isoformAK092666_02 Sequence ID: No. 185 Accession #: AK092666_04aa Sequencedefinition: Novel amino acids generated by spliced isoform AK092666_04

REFERENCES

-   Alcaraz et al., Cancer Res., 55:3998-4002, 1994.-   Allhoff et al., World J. Urol., 7:12-16, 1989.-   An et al., Proc. Amer. Assn. Canc. Res., 36:82, 1995.-   An et al., Molec. Urol., 2: 305-309, 1998.-   Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold    Spring Harbor Press, Cold Spring Harbor, N.Y., 1988.-   Babian et al., J. Urol., 156:432-437, 1996.-   Badalament et al., J. Urol., 156:1375-1380, 1996.-   Baichwal and Sugden, In: Gene Transfer, Kucherlapati (Ed.), Plenum    Press, New York, pp 117-148, 1986.-   Bangharn et al., J. Mol. Biol. 13: 238-252, 1965.-   Barinaga, Science, 271: 1233, 1996.-   Bedzyk et al., J. Biol. Chem., 265:18615, 1990-   Bell et al., “Gynecological and Genitourinary Tumors,” In:    Diagnostic Immunopathology, Colvin, Bhan and McCluskey (Eds.), 2nd    edition, Ch. 31, Raven Press, New York, pp 579-597, 1995.-   Bellus, J Macromol. Sci. Pure Appl. Chem., A31(1):1355-1376, 1994.-   Benvenisty and Neshif, Proc. Nat. Acad Sci. USA, 83:9551-9555, 1986.-   Bittner et al., Methods in Enzymol, 153:516-544, 1987.-   Bookstein et al., Science, 247:712-715, 1990a.-   Bookstein et al., Proc. Nat'l Acad. Sci. USA, 87:7762-7767, 1990b.-   Bova et al., Cancer Res., 53:3869-3873, 1993-   Brawn et al., The Prostate, 28:295-299, 1996.-   Campbell, In: Monoclonal Antibody Technology, Laboratory Techniques    in Biochemistry and Molecular Biology, Burden and Von Knippenberg    (Eds.), Vol.13:75-83, Elsevier, Amsterdam, 1984.-   Capaldi et al., Biochem. Biophys. Res. Comm, 76:425, 1977.-   Carter and Coffey, In: Prostate Cancer: The Second Tokyo    Symposium, J. P. Karr and H. Yamanak (Eds.), Elsevier, New York, pp    19-27, 1989.-   Carter and Coffey, Prostate, 16:3948, 1990.-   Carter et al., Proc. Nat'l Acad Sci. USA, 87:8751-8755, 1990.-   Carter et al., Proc. Nat'l Acad Sci. USA93: 749-753, 1996.-   Carter et al., J. Urol., 157:2206-2209, 1997.-   Cech et al., Cell, 27:487496, 1981.-   Chang et al., Hepatology, 14: 124A, 1991.-   Chaudhary et al., Proc. Nat'l Acad. Sci., 87:9491, 1990-   Chen and Okayama, MoL Cell Biol., 7:2745-2752, 1987.-   Chen et al., Clin. Chem., 41:273-282, 1995a.-   Chen et al., Proc. Am. Urol. Assn, 153:267A, 1995.-   Chinault and Carbon, “Overlap Hybridization Screening: Isolation and    Characterization of Overlapping DNA Fragments Surrounding the LEU2    Gene on Yeast Chromosome III,” Gene, 5:111-126, 1979.-   Chomczynski and Sacchi, Anal. Biochem., 162:156-159, 1987.-   Christensson et al., J. Urol., 150:100-105, 1993.-   Coffin, In: Virology, Fields et al. (Eds.), Raven Press, New York,    pp 1437-1500, 1990.-   Colberre-Garapin et al., J. Mol. Biol., 150:1, 1981.-   Colvin et al., Diagnostic Immunopathology, 2nd edition, Raven Press,    New York, 1995.-   Cooner et al., J. Urol., 143:1146-1154, 1990.-   Couch et al., Am. Rev. Resp. Dis., 88:394-403, 1963.-   Coupar et al., Gene, 68:1-10, 1988.-   Culver et al., Science, 256:1550-1552, 1992.-   Davey et al., EPO No. 329 822.-   Deamer and Uster, “Liposome Preparation: Methods and Mechanisms,”    In: Liposomes, M. Ostro (Ed.), 1983.-   Diamond et al., J. Urol., 128:729-734, 1982.-   Donahue et al., J. Biol. Chem., 269:8604-8609, 1994.-   Dong et al., Science, 268:884-886, 1995.-   Dubensky et al., Proc. Nat. Acad. Sci. USA, 81:7529-7533, 1984.-   Dumont et al., J. Immunol., 152:992-1003, 1994.-   Elledge et al., Cancer Res. 54:3752-3757, 1994-   European Patent Application EPO No. 320 308-   Fearon et al., Science, 247:47-56, 1990.-   Fechheirneret al., Proc. Natl. Acad. Sci. USA, 84:8463-8467, 1987.-   Forster and Symons, Cell, 49:211-220, 1987.-   Fraley et al., Proc. Natl. Acad. Sci USA, 76:3348-3352, 1979.-   Friedmann, Science, 244:1275-1281, 1989.-   Freifelder, In: Physical Biochemistry Applications to Biochemistry    and Molecular Biology, 2nd ed., Wm. Freeman and Co., New York, N.Y.,    1982.-   Frohman, In: PCR Protocols: A Guide to Methods and Applications,    Academic Press, New York, 1990.-   Gefter et al., Somatic Cell Genet., 3:231-236, 1977.-   Gerlach et al., Nature (London), 328:802-805, 1987.-   Ghosh-Choudhury et al., EMBO J., 6:1733-1739, 1987.-   Gingeras et al., PCT Application WO 88/10315.-   Ghosh and Bachhawat, In: Liver Diseases, Targeted Diagnosis and    Therapy Using Specific Receptors and Ligands, Wu et al. (Eds.),    Marcel Dekker, New York, pp 87-104, 1991.-   Goding, In: Monoclonal Antibodies: Principles and Practice, 2nd ed.,    Academic Press, Orlando, Fla., pp 60-61, 65-66, 71-74, 1986.-   Gomez-Foix et al., J. Biol. Chem., 267:25129-25134, 1992.-   Gopal, Mol. Cell Biol., 5:1188-1190, 1985.-   Graham et al., J. Gen. Virol., 36:59-72, 1977.-   Graham and van der Eb, Virology, 52:456-467, 1973.-   Graham and Prevec, In: Methods in Molecular Biology: Gene Transfer    and Expression Protocols 7,-   E. J. Murray (Ed.), Humana Press, Clifton, N.J., pp 205-225, 1991.-   Gregoriadis (ed.), In: Drug Carriers in Biology and Medicine, pp    287-341, 1979.-   Grunhaus and Horwitz, Sem. Virol., 3:237-252, 1992.-   Harland and Weintraub, J. Cell Biol., 101:1094-1099, 1985.-   Harris et al., J. Urol., 157:1740-1743, 1997.-   Heng et al., Proc. Nat. Acad. Sci. USA, 89: 9509-9513, 1992.-   Hermonat and Muzycska, Proc. Nat. Acad. Sci USA, 81:6466-6470, 1984.-   Hersdorffer et al., DNA Cell Biol., 9:713-723, 1990.-   Herz and Gerard, Proc. Natl Acad Sci. USA, 90:2812-2816, 1993.-   Hess et al., J. Adv. Enzyme Reg., 7:149, 1968.-   Hitzeman et al., J. Biol. Chem., 255:2073, 1980.-   Holland et al., Biochemistry, 17:4900, 1978.-   Horoszewicz, Kawinski and Murphy, Anticancer Res., 7:927-936, 1987.-   Horwich, et al., J. Virol., 64:642-650, 1990.-   Huang et al., Prostate, 23: 201-212, 1993.-   Innis et al., In: PCR Protocols, Academic Press, Inc., San Diego    Calif., 1990.-   Inouye et al., Nucl. Acids Res., 13:3101-3109, 1985.-   Isaacs et al., Cancer Res., 51:4716-4720, 1991.-   Isaacs et al., Sem. Oncol., 21:1-18, 1994.-   Israeli et al., Cancer Res., 54:1807-1811, 1994.-   Jacobson et al., JAMA, 274:1445-1449, 1995.-   Johnson et al., In: Biotechnology and Pharmacy, Pezzuto et al.,    (Eds.), Chapman and Hall, New York, 1993.-   Jones, Genetics, 85:12, 1977.-   Jones and Shenk, Cell, 13:181-188, 1978.-   Joyce, Nature, 338:217-244, 1989.-   Kaneda et al., Science, 243:375-378, 1989.-   Kato el al., J. Biol. Chem., 266:3361-3364, 1991.-   Kent, et al., Genome Res. 12:996-1006 (2002).-   Kim and Cech, Proc. Natl. Acad. Sci. USA, 84:8788-8792, 1987.-   Kingsman el al., Gene, 7:141, 1979.-   Klein et al., Nature, 327:70-73, 1987.-   Kohler and Milstein, Nature, 256:495-497, 1975.-   Kohler and Milstein, Eur. J. Immunol., 6:511-519, 1976.-   Kwoh et al., Proc. Nat. Acad. Sci. USA, 86:1173, 1989.-   Landis et al., CA Cancer J. Clin., 48: 6-29, 1998.-   Le Gal La Salle et al., Science, 259:988-990, 1993.-   Levrero et al., Gene, 10 1: 195-202, 1991.-   Liang and Pardee, Science, 257:967-971, 1992.-   Liang and Pardee, U.S. Pat. No. 5,262,311, 1993.-   Liang et al., Cancer Res., 52:6966-6968, 1992.-   Lifton, Science, 272:676, 1996.-   Lilja et al., Clin. Chem., 37:1618-1625, 1991.-   Lithrup et al., Cancer, 74:3146-3150, 1994.-   Lowy et al., Cell, 22:817, 1980.-   Macoska et al., Cancer Res., 54:3824-3830, 1994.-   Mann et al., Cell, 33:153-159, 1983.-   Markowitz et al., J. Virol., 62:1120-1124, 1988.-   Marley et al., Urology, 48(6A): 16-22, 1996.-   McCormack et al., Urology, 45:729-744, 1995.-   Michel and Westhof, J. Mol. Biol. 216:585-610, 1990.-   Miki et al., Science, 266:66-71, 1994.-   Miller et al., PCT Application, WO 89/06700.-   Mok et al., Gynecol. Oncol., 52:247-252, 1994.-   Morahan et al., Science 272:1811, 1996.-   Mulligan et al., Proc. Nat'l Acad. Sci. USA, 78:2072, 1981.-   Mulligan, Science, 260:926-932, 1993.-   Murphy et al., Cancer, 78: 809-818, 1996.-   Murphy et al., Prostate, 26:164-168, 1995.-   Nakamura et al., In: Handbook of Experimental Immunology, (4th Ed.),    Weir, E., Herzenberg, L. A.; Blackwell, C., Herzenberg, L. (Eds.),    Vol. 1, Chapter 27, Blackwell Scientific Publ., Oxford, 1987.-   Nicolas and Rubinstein, In: Vectors: A Survey of Molecular Cloning    Vectors and Their Uses, Rodriguez and Denhardt (Eds.), Butterworth,    Stoneham, p 494-513, 1988.-   Nicolau and Sene, Biochim. Biophys. Acta, 721:185-190, 1982.-   O'Dowd et al., J. Urol., 158:687-698, 1997.-   O'Hare et al., Proc. Nat'l Acad. Sci. USA, 78:1527, 1981.-   Oesterling et al., J. Urol., 154:1090-1095, 1995.-   Ohara et al., Proc. Nat'l Acad. Sci. USA, 86:5673-5677, 1989.-   Orozco et al., Urology, 51:186-195, 1998.-   Parker et al., CA Cancer J. Clin., 65:5-27, 1996.-   Partin and Oesterling, Urology, 48 (6A): 1-3, 1996.-   Partin and Oesterling, J. Urol., 152:1358-1368, 1994.-   Partin and Oesterling (Eds.), Urology, 48(6A) Supplement: 1-87,    1996.-   Paskind et al., Virology, 67:242-248, 1975.-   PCT Application No. PCT/US87/00880-   Pettersson et al., Clin. Chem., 41(10):1480-1488, 1995.-   Piironen et al., Clin. Chem. 42:1034-1041, 1996.-   Potter et al., Proc. Nat. Acad. Sci. USA, 81:7161-7165, 1984.-   Racher et al., Biotechnology Techniques, 9:169-174, 1995.-   Ragot et al., Nature, 361:647-650, 1993.-   Ralph and Veltri, Advanced Laboratory, 6:51-56, 1997.-   Ralph et al., Proc. Natl. Acad. Sci. USA, 90(22):10710-10714, 1993.-   Reinhold-Hurek and Shub, Nature, 357:173-176, 1992.-   Renan, Radiother. Oncol., 19:197-218, 1990.-   Ribas de Pouplana and Fothergill-Gilmore, Biochemistry,    33:7047-7055, 1994.-   Rich et al., Hum. Gene Ther., 4:461-476, 1993.-   Ridgeway, In: Vectors: A Survey of Molecular Cloning Vectors and    Their Uses, Rodriguez R L, Denhardt D T (Eds.), Butterworth,    Stoneham, pp 467492, 1988.-   Rippe et al., Mol. Cell Biol., 10:689-695, 1990.-   Rosenfeld et al., Science, 252:431-434, 1991.-   Rosenfeld et al., Cell, 68:143-155, 1992.-   Roux et al., Proc. Nat'l Acad. Sci. USA, 86:9079-9083, 1989.-   Sager et al., FASEB J., 7:964-970, 1993.-   Sambrook et al., (ed.), In: Molecular Cloning, Cold Spring Harbor    Laboratory Press, Cold Spring Harbor, N.Y., 1989.-   Santerre et al., Gene, 30: 147-156, 1984.-   Sarver, et al., Science, 247:1222-1225, 1990.-   Scanlon et al., Proc Natl Acad Sci USA, 88:10591-10595, 1991.-   Sidransky et al., Science, 252:706-709, 1991.-   Sidransky et al., Cancer Res., 52:2984-2986, 1992.-   Silver et al., Clin. Cancer Res., 3:81-85, 1997.-   Slamon et al., Science, 224:256-262, 1984.-   Slamon et al., Science, 235:177-182, 1987.-   Slamon et al., Science, 244:707-712, 1989.-   Smith, U.S. Pat. No. 4,215,051.-   Soh et al., J. Urol., 157:2212-2218, 1997.-   Stenman et al., Cancer Res., 51:222-226, 1991.-   Stinchcomb et al., Nature, 282:39, 1979.-   Stratford-Perricaudet and Perricaudet, In: Human Gene Transfer, O.    Cohen-Haguenauer et al., (Eds.), John Libbey Eurotext, France, pp    51-61, 1991.-   Stratford-Perricaudet et al., Hum. Gene. Ther., 1:241-256, 1990.-   Sun and Cohen, Gene, 137:127-132, 1993.-   Szoka and Papahadjopoulos, Proc. Nat'l. Acad. Sci. USA, 75:    4194-4198, 1978.-   Szybalska et al., Proc. Nat'l Acad. Sci. USA, 48:2026, 1962.-   Takahashi et al., Cancer Res., 54:3574-3579, 1994.-   Taparowsky et al., Nature, 300:762-764, 1982.-   Temin, In: Gene Transfer, Kucherlapati R. (Ed.), Plenum Press, New    York, pp 149-188:, 1986.-   Tooze, In: Molecular Biology of DNA Tumor Viruses, 2nd ed., Cold    Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1991.-   Top et al., J. Infect. Dis., 124:155-160, 1971.-   Tschemper et al., Gene, 10:1 57, 1980.-   Tur-Kaspaet al., Mol. Cell Biol., 6:716-718, 1986.-   U.S. patent application Ser. No. 08/692,787-   U.S. Pat. No. 4,196,265-   U.S. Pat. No. 4,215,051-   U.S. Pat. No. 4,683,195-   U.S. Pat. No. 4,683,202-   U.S. Pat. No. 4,800,159-   U.S. Pat. No. 4,883,750-   U.S. Pat. No. 5,354,855-   U.S. Pat. No. 5,359,046-   Varmus et al., Cell, 25:23-36, 1981.-   Veltri et al., J. Cell Biochem., 19(suppl):249-258, 1994.-   Veltri et al., Urology, 48: 685-691, 1996.-   Veltri et al., Sem. Urol. Oncol., 16:106-117, 1998.-   Veltri et al., Urology,53:139-147, 1999.-   Visakorpi et al., Am. J. Pathol., 145:1-7, 1994.-   Wagner et al., Science, 260:1510-1513, 1993.-   Walker et al., Proc. Nat'l Acad. Sci. USA, 89:392-396, 1992.-   Watson et al., Cancer Res., 54:4598-4602, 1994.-   Welsh et al., Nucl. Acids Res., 20:4965-4970, 1992.-   Wigler et at, Cell, 11:223, 1977.-   Wigler et al., Proc. Nat'l Acad. Sci. USA, 77:3567, 1980.-   Wingo et al., CA Cancer J. Clin., 47: 239-242, 1997.-   WO 90/07641, filed Dec. 21, 1990.-   Wong et al., Int. J. Oncol., 3:13-17, 1993.-   Wu and Wu, J. Biol. Chem., 262: 4429-4432, 1987.-   Wu and Wu, Biochemistry, 27: 887-892, 1988.-   Wu and Wu, Adv. Drug Delivery Rev., 12: 159-167, 1993.-   Wu el al., Genomics, 4:560, 1989.-   Yang et al., Proc. Natl. Acad. Sci. USA, 87:9568-9572, 1990.-   Yokoda et al., Cancer Res. 52, 3402-3408, 1992.-   Zlotta et al, J. Urol., 157:1315-1321, 1997.

1. An isolated nucleic acid sequence that is expressed by human prostatecancer cells, selected from the group consisting of: (i) the nucleicacid sequence contained in SEQ ID NOS.: 1 to 173, 175, 177, 179, 181;(ii) variants thereof, wherein such variants have a nucleic acidsequence that is at least 70% identical to the sequence of (i) whenaligned without allowing for gaps; and (iii) fragments of (i) or (ii)having a size of at least 20 nucleotides in length.
 2. The nucleic acidsequence of claim 1 which comprises the nucleic acid sequence containedin any one of SEQ ID NOS.: 1 to 173, 175, 177, 179, 181 or a fragmentthereof.
 3. A primer mixture that comprises primers that result in thespecific amplification of one of the nucleic acid sequences of claim 1.4. A method of detecting prostate cancer comprising determining whethera human prostate cell sample expresses a target nucleic acid molecule,wherein said target nucleic acid molecule comprises the sequence of agene or RNA comprising a nucleic acid sequence selected from the groupconsisting of SEQ ID NOS.: 1 to 173, 175, 177, 179, 181 or of a fragmentof said gene or RNA having a size of at least 20 nucleotides in length.5. The method of claim 4, wherein said method comprises detecting theexpression of said target nucleic acid molecule using a nucleic acidsequence that specifically hybridizes thereto.
 6. The method of claim 5,wherein said method comprises detecting the expression of said targetnucleic acid molecule using primers that result in the amplificationthereof.
 7. The method of claim 5, wherein the expression of said targetnucleic acid molecule is detected by assaying for the antigen encoded bysaid nucleic acid.
 8. The method of claim 7, wherein said assay involvesthe use of a monoclonal antibody or fragment that specifically binds tosaid antigen.
 9. The method of claim 8, wherein said assay comprises anELISA or competitive binding assay.
 10. An antigen expressed by humanprostate cancer cells, wherein said antigen is selected from the groupconsisting of: (i) the antigen encoded by a nucleic acid sequence havingat least 90% sequence identity in SEQ ID NOS.: 1 to 173, 175, 177, 179,181; (ii) an antigen derived from a protein comprising a sequenceshaving at least 90% identity in SEQ ID NOS. 174, 176, 178, 180, 182-185;and (iii) an antigenic fragment of (i) or (ii).
 11. A prostate antigencomprising (i) the amino acid sequence encoded by a nucleic acidsequence selected from the group consisting of SEQ ID NOs.: 1 to 173,175, 177, 179, 181 or (ii) an amino acid sequence selected from SEQ IDNOS.: 174, 176, 178, 180, and 182-185, or (iii)an antigenic fragment of(i) or (ii).
 12. A monoclonal antibody or antigen-binding fragmentthereof that specifically binds to a target polypeptide moleculeselected from: (i) a polypeptide encoded by a nucleic acid moleculecomprising the sequence of a gene or RNA comprising a sequence selectedfrom the group consisting of SEQ ID NOS.: 1 to 173, 175, 177, 179, 181,or by a fragment of said gene or RNA having a size of at least 20nucleotides in length, or a polypeptide derived from SEQ ID NOS. :174,176, 178, 180, and 182-185 (ii) an antigen according to claim 10, and(iii) an antigenic fragment of (i) or (ii).
 13. A monoclonal antibody orfragment thereof that specifically binds the antigen of claim
 11. 14.The antigen of claim 10 which is attached directly or indirectly to adetectable label.
 15. The antibody of claim 12 which is attacheddirectly or indirectly to a detectable label.
 16. A diagnostic kit fordetection of prostate cancer which comprises a DNA according to claim 1and a detectable label.
 17. A diagnostic kit for detection of prostatecancer which comprises primers according to claim 3 and a diagnosticallyacceptable carrier.
 18. A diagnostic kit for detection of prostatecancer which comprises a monoclonal antibody according to claim 12 and adetectable label.
 19. A method for treating prostate cancer, whichcomprises administering to a subject a therapeutically effective amountof a ligand which specifically binds a target molecule selected from (i)a gene or RNA comprising a sequence selected from the group consistingof SEQ ID NOS.: 1 to 173, 175, 177, 179, 181, a variant thereof or afragment of said gene or RNA having a size of at least 20 nucleotides inlength, and (ii) a protein or polypeptide encoded by a gene or RNAcomprising a sequence selected from the group consisting of SEQ ID NOS.:1 to 173, 175, 177, 179, 181, a variant thereof or a fragment of saidgene or RNA having a size of at least 20 nucleotides in length, or apolypeptide derived from SEQ ID NOS.:174, 176, 178, 180, and 182-185.20. The method of claim 19, wherein the ligand is a ribozyme orantisense oligonucleotide that inhibits the expression of a gene havinga DNA sequence selected from the group consisting of SEQ ID NOS.: 1 to173, 175, 177, 179, 181or a fragment, or variant thereof, or apolypeptide derived from SEQ ID NOS.:174, 176, 178, 180, and 182-185.21. The method of claim 19, wherein the ligand is directly or indirectlyattached to an effector moiety.
 22. The method of claim 21, wherein saideffector moiety is a therapeutic radiolabel, enzyme, cytotoxin, growthfactor, or drug.
 23. A method for treating prostate cancer comprisingadministering to a subject a therapeutically effective amount of anantigen according to claim 10, and optionally an adjuvant that elicits ahumoral or cytotoxic T-lymphocyte response to said antigen.
 24. A methodfor treating prostate cancer comprising administering to a subject atherapeutically effective amount of a ligand which specifically binds toa protein encoded by a gene or RNA comprising a sequence selected fromthe group consisting of SEQ ID NOS.: 1 to 173, 175, 177, 179, 181 or afragment, or variant thereof, or a polypeptide derived from SEQ IDNOS.:174, 176, 178, 180, and 182-185 optionally directly or indirectlyattached to a therapeutic effector moiety.
 25. The method of claim 24,wherein said effector moiety is a radiolabel, enzyme, cytotoxin, growthfactor, or drug.
 26. The method of claim 25 wherein the radiolabel isyttrium.
 27. The method of claim 25 wherein the radiolabel is indium.28. The method of claim 24 wherein said ligand is a monoclonal antibodyor fragment thereof.
 29. The method of claim 24 wherein said ligand is asmall molecule.
 30. The method of claim 24 wherein said ligand is apeptide.
 31. The method of claim 24, wherein said ligand binds anextracellular domain of said protein.
 32. A molecule, selected from: (i)a polypeptide comprising the sequence of an extracellular domain of aprotein encoded by a gene or RNA comprising a sequence selected from thegroup consisting of SEQ ID NOS.: 1 to 185; and (ii) a nucleic acidmolecule encoding a polypeptide of (i).
 33. The molecule of claim 32,wherein said polypeptide has 8 to 100 amino acids in length.
 34. Amethod for selecting, identifying, screening, characterizing oroptimizing biologically active compounds, comprising contacting acandidate compound with a target molecule and determining whether thecandidate compound binds said target molecule, wherein said targetmolecule is selected from (i) a nucleic acid molecule comprising thesequence of a gene or RNA comprising a nucleic acid sequence selectedfrom the group consisting of SEQ ID NOS.: 1 to 173, 175, 177, 179, 181,(ii) a fragment of said gene or RNA having a size of at least 20nucleotides in length, and (iii) a polypeptide encoded by (i) or (ii) ora polypeptide derived from SEQ ID NOs.:174, 176, 178, 180, and 182-185.